Most Sage customers use their Pippins with an Illumina workflow, so for the next few weeks we’ll be taking a look at some of the most common or interesting applications of automated DNA sizing with the HiSeq, MiSeq, and even the GA II or GA IIx workhorses.
Today we look at mate-pair sequencing, a protocol using a large insert between reads to cover greater genomic distances. The approach is used to span highly repetitive regions and can result in longer contigs and fewer assembly gaps. Often, mate-pair sequence data is combined with shorter-insert paired-end reads to cover a genome more thoroughly. It is used for de novo sequencing as well as structural variant detection and genome finishing.
For Illumina sequencing, the Pippin Prep and BluePippin save time and provide a reproducible alternative to manual size selection that will ensure better sequencing results. Mate-pair sequencing can be tricky, so the more precise your library sizing, the more accurate your data will be in the end. If you’re performing the newer mate-pair sequencing with Nextera, Illumina recommends using the Pippin platform to get “more stringent” sizing than can be accomplished with AMPure alone. (We’re under Size Selection in Chapter 3, beginning on page 40 of the guide.) The document reports that “in our experience running a standard agarose gel does not provide as robust and reproducible results as the Sage Pippin Prep.” We’re honored to be in the official guidelines!
A paper from scientists at the Wellcome Trust Sanger Institute last year offers another take on the mate-pair protocol. The publication, “An improved approach to mate-paired library preparation for Illumina sequencing,” describes optimized techniques to boost library complexity and quality while reducing the occurrence of chimeras. The technique was designed to improve mate-pair success when not much DNA is available or the sample is degraded. BluePippin is a critical part of the final workflow. While the authors noted that other size selection methods could be used, they note, “The Blue Pippin provides the greatest recovery and accuracy of currently available commercial methods.”
Mate-pair sequencing is a great fit for our newest instrument, the SageELF, which performs whole-sample fractionation. SageELF generates 12 contiguous fractions from a DNA sample, allowing scientists to build short-insert and long-insert libraries from the same sample.
Our blog series will continue with peeks into how scientists are using Pippin and Illumina sequencers together for microRNA studies, ChIP-seq, ddRADseq, and more. Check back soon for more!