2nd generation sequencing
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The Roche/454 Life Sciences HTS process begins with .7 μg of any double-stranded starting material (DNA from soil, water, plant, animal, environmental, etc). High-molecular weight DNA is preferred (1.8 OD, 1.5 kb pieces or larger) for genomic projects. Following an accurate quantification of the DNA concentration by a fluorescence method (i.e., PicoGreen), the DNA is fractionated into a size-range conducive for emulsion PCR (emPCR) and subsequent pyrosequencing. Nebulized DNA is then filled in (=blunted) prior to library preparation.
Whether the focus is viruses in a water sample, diversity of methanotrophs in a sediment sample, or characterizing a de novo genome of an organism, high-throughput sequencing (HTS) has fundamentally changed the way questions in metagenomics are approached.
About 2nd Generation Sequencing
HTS provides researchers and scientists with a revolutionary tool for quickly and affordably obtaining DNA sequence. The cost per unit of DNA sequence (i.e., base pair) obtained with HTS compared to traditional Sanger sequencing is dramatically more cost and time effective.
The Roche 454 Advantage
- It is the only platform that allows for long reads with reads of between 400 and 500 million base pairs with Titanium chemistry.
- The Roche 454 platform is the most popular to use for de novo sequencing.
- It is the most reliable of the three major platforms as measured by a recent In Sequence Survey.