{"id":1218,"date":"2022-08-05T19:25:02","date_gmt":"2022-08-05T13:40:02","guid":{"rendered":"https:\/\/microbenotes.com\/?p=1218"},"modified":"2023-02-11T16:23:09","modified_gmt":"2023-02-11T10:38:09","slug":"next-generation-sequencing-ngs","status":"publish","type":"post","link":"https:\/\/microbenotes.com\/next-generation-sequencing-ngs\/","title":{"rendered":"Next-Generation Sequencing (NGS)- Definition, Types"},"content":{"rendered":"\n
Next Generation Sequencing (NGS) is a robust platform that has enabled the sequencing of thousands to millions of DNA<\/a> molecules simultaneously.<\/strong><\/span><\/p>\n\n\n\nNext-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies.<\/span><\/p>\n\n\n\n<\/figure>\n\n\n\nThe high demand for low-cost sequencing has driven the development of high-throughput sequencing,<\/span> which produces thousands or millions of sequences at once.<\/span><\/p>\n\n\n\nThey are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.<\/span><\/p>\n\n\n\nThus, these recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and as such, have revolutionized the study of genomics and molecular biology.<\/span><\/p>\n\n\n\nClassified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies.<\/span><\/p>\n\n\n\n<\/figure>\n\n\n\nThe high demand for low-cost sequencing has driven the development of high-throughput sequencing,<\/span> which produces thousands or millions of sequences at once.<\/span><\/p>\n\n\n\nThey are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.<\/span><\/p>\n\n\n\nThus, these recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and as such, have revolutionized the study of genomics and molecular biology.<\/span><\/p>\n\n\n\nClassified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
The high demand for low-cost sequencing has driven the development of high-throughput sequencing,<\/span> which produces thousands or millions of sequences at once.<\/span><\/p>\n\n\n\nThey are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.<\/span><\/p>\n\n\n\nThus, these recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and as such, have revolutionized the study of genomics and molecular biology.<\/span><\/p>\n\n\n\nClassified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
They are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.<\/span><\/p>\n\n\n\nThus, these recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and as such, have revolutionized the study of genomics and molecular biology.<\/span><\/p>\n\n\n\nClassified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Thus, these recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing and as such, have revolutionized the study of genomics and molecular biology.<\/span><\/p>\n\n\n\nClassified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Classified to different generations, NGS has led to overcoming the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. <\/span><\/p>\n\n\n\nNext-Generation Sequencing (NGS) Techniques<\/figcaption><\/figure>\n\n\n\nThe generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
The generations it is classified into include:<\/span><\/p>\n\n\n\nFirst Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
First Generation <\/strong><\/span><\/p>\n\n\n\n\nSanger Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nSecond Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Second Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nPyrosequencing<\/span><\/li>\n\n\n\nSequencing by Reversible Terminator Chemistry<\/span><\/li>\n\n\n\nSequencing by Ligation<\/span><\/li>\n<\/ul>\n\n\n\n Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Third Generation Sequencing <\/strong><\/span><\/p>\n\n\n\n\nSingle Molecule Fluorescent Sequencing<\/span><\/li>\n\n\n\nSingle Molecule Real Time Sequencing<\/span><\/li>\n\n\n\nSemiconductor Sequencing<\/span><\/li>\n\n\n\nNanopore Sequencing<\/span><\/li>\n<\/ul>\n\n\n\nFourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Fourth Generation Sequencing<\/strong><\/span><\/p>\n\n\n\nAims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Aims conducting genomic analysis directly in the cell.<\/span><\/p>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\n\nTable of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
Table of Contents<\/p>\nToggle<\/span><\/path><\/svg><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\nNext-Generation Sequencing Types<\/a><\/li>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/a><\/li>Polony sequencing<\/a><\/li>Pyrosequencing<\/a><\/li>Illumina (Solexa) sequencing<\/a><\/li>SOLiD sequencing<\/a><\/li>DNA nanoball sequencing<\/a><\/li>Helioscope single molecule sequencing<\/a><\/li>Single molecule SMRT sequencing<\/a><\/li>Single molecule real time (RNAP) sequencing<\/a><\/li>References<\/a><\/li><\/ul><\/nav><\/div>\n<\/span>Next-Generation Sequencing Types<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/span>Lynx therapeutics’ massively parallel signature sequencing (MPSS)<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\nIt is considered as the first of the “next-generation” sequencing technologies.<\/span><\/li>\n\n\n\nMPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr.<\/span><\/li>\n\n\n\nMPSS is an ultra high throughput sequencing technology.<\/span><\/li>\n\n\n\nWhen applied to expression profile, it reveals almost every transcript in the sample and provide its accurate expression level.<\/span><\/li>\n\n\n\nMPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.<\/span><\/li>\n\n\n\nHowever, the essential properties of the MPSS output were typical of later “next-gen” data types, including hundreds of thousands of short DNA sequences.<\/span><\/li>\n\n\n\nIn the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Polony sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\nIt is an inexpensive but highly accurate multiplex sequencing technique that can be used to read millions of immobilized DNA sequences in parallel.<\/span><\/li>\n\n\n\nThis technique was first developed by Dr. George Church in Harvard Medical college.<\/span><\/li>\n\n\n\nIt combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1\/10 that of Sanger sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Pyrosequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nPyrosequencing<\/figcaption><\/figure>\n\n\n\n\nA parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics.<\/span><\/li>\n\n\n\nThe method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.<\/span><\/li>\n\n\n\nThe sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.<\/span><\/li>\n\n\n\nPyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.<\/span><\/li>\n\n\n\nThis technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Illumina (Solexa) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\nIllumina sequencing<\/figcaption><\/figure>\n\n\n\n\nSolexa developed a sequencing technology based on dye terminators.<\/span><\/li>\n\n\n\nIn this method, DNA molecule are first attached to primers on a slide and amplified. This is known as bridge amplification.<\/span><\/li>\n\n\n\nUnlike pyrosequencing, the DNA can only be extended one nucleotide at a time.<\/span><\/li>\n\n\n\nA camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3′ blocker is chemically removed from the DNA, allowing the next cycle to commence.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>SOLiD sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\nThe technology for sequencing used in ABISolid sequencing is oligonucleotide ligation and detection.<\/span><\/li>\n\n\n\nIn this, a pool of all possible oligonucleotides of fixed length are labelled according to the sequenced position.<\/span><\/li>\n\n\n\nThis sequencing results to the sequences of quantities and lengths comparable to illumine sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>DNA nanoball sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\nIt is high throughput sequencing technology that is used to determine the entire genomic sequence of an organism.<\/span><\/li>\n\n\n\nThe method uses rolling circle replication to amplify fragments of genomic DNA molecules.<\/span><\/li>\n\n\n\nThis DNA sequencing allows large number of DNA nanoballs to be sequenced per run and at low reagent cost compared to other next generation sequencing platforms.<\/span><\/li>\n\n\n\nHowever, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult.<\/span><\/li>\n\n\n\nThis technology has been used for multiple genome sequencing projects and is scheduled to be used for more.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Helioscope single molecule sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n\nHelioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.<\/span><\/li>\n\n\n\nThe next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides.<\/span><\/li>\n\n\n\nThe reads are performed by the Helioscope sequencer.<\/span><\/li>\n\n\n\nThe reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers and RNA sequencing.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule SMRT sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n\nSMRT sequencing is based on the sequencing by synthesis approach.<\/span><\/li>\n\n\n\nThe DNA is synthesisd in so called zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well.<\/span><\/li>\n\n\n\nThe sequencing is performed with use of unmodified polymerase and fluorescently labelled nucleotides flowing freely in the solution.<\/span><\/li>\n\n\n\nThe wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.<\/span><\/li>\n\n\n\nThe fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.<\/span><\/li>\n\n\n\nThe SMTR technology allows detection of nucleotide modifications. This happens through the observation of polymerase kinetics.<\/span><\/li>\n\n\n\nThis approach allows reads of 1000 nucleotides.<\/span><\/li>\n<\/ul>\n\n\n\n<\/span>Single molecule real time (RNAP) sequencing<\/strong><\/span><\/span><\/h2>\n\n\n\n<\/figure><\/div>\n\n\n<\/span><\/p>\n\n\n\n
<\/span><\/p>\n\n\n\n