Scientists Optimize Mate-Pair Sequencing with SageELF

In a Biotechniques paper this month, scientists from The Genome Analysis Centre describe a new method for mate-pair sequencing that saves time and money while decreasing the amount of input DNA required. The method is based on SageELF, which automatically generates 12 contiguous fractions of DNA from a single sample.

Led by Darren Heavens, the authors report that length and quantity of input DNA have been problematic factors in the preparation of long mate-pair (LMP) libraries for next-gen sequencing. To address that issue, they adjusted the sample prep protocol to use SageELF instead of conventional gel-based sizing, and then chose the fraction that best met their target fragment length.

“Using the SageELF streamlines the library construction process, allowing LMP libraries >10 kb to be constructed in under 2 days with <10 µg input material,” the scientists write. “For many genome projects, multiple insert size LMP libraries are required, and the ability to construct up to 12 discretely sized libraries for a combined reagent cost of $1270 compared with the reagent cost of $715 for a single insert size LMP library highlights the potential cost savings.” The protocol was developed to optimize the Nextera-based long mate-pair kit for library construction. In addition to the initial round of size selection with SageELF, the scientists conduct another sizing step on the BluePippin prior to Illumina sequencing to ensure selection of DNA fragments best suited for the platform. The protocol pays off by saving time and money in library prep, as well as by reducing the need for larger volumes of input DNA. It also leads to better sequencing results. “Accurately determining the size and span of the inserts for mate pair libraries simplifies the scaffolding problem, enabling the assembly of longer, more precise sequences with fewer non-determined bases (runs of N bases), empowering all subsequent downstream analysis,” the scientists report. Check out the full paper: “A method to simultaneously construct up to 12 differently sized Illumina Nextera long mate pair libraries with reduced DNA input, time, and cost.”

And for more on the TGAC team, check out this brief profile.

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