A High-Throughput Method for Illumina RNA-Seq Library Preparation
Identifieur interne : 000537 ( Main/Exploration ); précédent : 000536; suivant : 000538A High-Throughput Method for Illumina RNA-Seq Library Preparation
Auteurs : Ravi Kumar [États-Unis] ; Yasunori Ichihashi [États-Unis] ; Seisuke Kimura [États-Unis] ; Daniel H. Chitwood [États-Unis] ; Lauren R. Headland [États-Unis] ; Jie Peng [États-Unis] ; Julin N. Maloof [États-Unis] ; Neelima R. Sinha [États-Unis]Source :
- Frontiers in Plant Science [ 1664-462X ] ; 2012.
Abstract
With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.
Url:
DOI: 10.3389/fpls.2012.00202
PubMed: 22973283
PubMed Central: 3428589
Affiliations:
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Headland</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Department of Plant Biology, University of California</institution><country>Davis, CA, USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea></wicri:regionArea><wicri:regionArea># see nlm:aff region in country</wicri:regionArea></affiliation></author><author><name sortKey="Peng, Jie" sort="Peng, Jie" uniqKey="Peng J" first="Jie" last="Peng">Jie Peng</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Department of Statistics, University of California</institution><country>Davis, CA, USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea></wicri:regionArea><wicri:regionArea># see nlm:aff region in country</wicri:regionArea></affiliation></author><author><name sortKey="Maloof, Julin N" sort="Maloof, Julin N" uniqKey="Maloof J" first="Julin N." last="Maloof">Julin N. Maloof</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Department of Plant Biology, University of California</institution><country>Davis, CA, USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea></wicri:regionArea><wicri:regionArea># see nlm:aff region in country</wicri:regionArea></affiliation></author><author><name sortKey="Sinha, Neelima R" sort="Sinha, Neelima R" uniqKey="Sinha N" first="Neelima R." last="Sinha">Neelima R. Sinha</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Department of Plant Biology, University of California</institution><country>Davis, CA, USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea></wicri:regionArea><wicri:regionArea># see nlm:aff region in country</wicri:regionArea></affiliation></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Benjamini, Y" uniqKey="Benjamini Y">Y. Benjamini</name></author><author><name sortKey="Hochberg, Y" uniqKey="Hochberg Y">Y. Hochberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Boutros, P C" uniqKey="Boutros P">P. C. Boutros</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Craig, D W" uniqKey="Craig D">D. W. Craig</name></author><author><name sortKey="Pearson, J V" uniqKey="Pearson J">J. V. Pearson</name></author><author><name sortKey="Szelinger, S" uniqKey="Szelinger S">S. 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Headland</name><name sortKey="Ichihashi, Yasunori" sort="Ichihashi, Yasunori" uniqKey="Ichihashi Y" first="Yasunori" last="Ichihashi">Yasunori Ichihashi</name><name sortKey="Kimura, Seisuke" sort="Kimura, Seisuke" uniqKey="Kimura S" first="Seisuke" last="Kimura">Seisuke Kimura</name><name sortKey="Maloof, Julin N" sort="Maloof, Julin N" uniqKey="Maloof J" first="Julin N." last="Maloof">Julin N. Maloof</name><name sortKey="Peng, Jie" sort="Peng, Jie" uniqKey="Peng J" first="Jie" last="Peng">Jie Peng</name><name sortKey="Sinha, Neelima R" sort="Sinha, Neelima R" uniqKey="Sinha N" first="Neelima R." last="Sinha">Neelima R. Sinha</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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