Scientists at the University of Arizona, led by senior author and newly named Moore Foundation Investigator Matthew Sullivan, have published details of how to optimize sample prep methods for next-gen sequencing projects in which input DNA is negligible. As part of that work, the authors recommend the Pippin platform from Sage Science as the superior technology for automated size selection of libraries.
The paper, “Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method,” from Duhaime et al., was published in September 2012 in Environmental Microbiology and is available via open access. The work focused on ways to reduce amplification bias, including precise DNA size selection, enzyme choice, and optimizing cycle number.
Sullivan and his team are using these methods for ocean-based viral metagenomics projects, but the methods translate to other projects that have very little DNA available. In this work, they looked at size selection in particular as a way to control size-related bias in the amplification step of sample preparation.
The paper reports on a comparison of Pippin to Solid Phase Reversible Immobilization (SPRI) from Beckman Coulter Genomics and to manual gels. The authors write:
“Of the three sizing fractionation methods tested for target recovery efficiency (fraction recovered DNA in target 400–600 bp size range), throughput (ease of applicability to numerous samples simultaneously), and risk of cross-sample contamination, Pippin Prep, an automated optical electrophoretic system that does not require gel extraction, was the most efficient and reproducible (94–96% of input DNA), with the tightest, most specific sizing.”
They note that SPRI was also high-throughput with low risk of contamination, but was the least efficient in recovery of the three methods tested, yielding 46-50 percent of the targeted size fraction after shearing.
“Based on this comparative analysis, we recommend the Pippin Prep automated electrophoretic system to prepare samples for [next-gen sequencing] libraries,” they conclude.
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