Unlocking short read sequencing for metagenomics.

Rodrigue S, Materna AC, Timberlake SC, Blackburn MC, Malmstrom RR, Alm EJ, Chisholm SW

BACKGROUND: Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved. METHODOLOGY/PRINCIPAL FINDINGS: We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read. CONCLUSIONS/SIGNIFICANCE: This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.

DNA shearing

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January, 2010



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