New Structural Variant Analysis Method Uses Targeted Capture and Sequencing of Large-Insert DNA

In a new BMC Genomics paper, scientists from Baylor College of Medicine describe a new method for accurate, affordable interrogation of structural variants across the human genome. We’re delighted to see that automated DNA size selection tools from Sage Science contributed to this important approach.

In the paper, lead authors Min Wang and Christine Beck, along with collaborators from Baylor’s genome center, cite the need for a method like this based on the difficulties of using next-gen sequencing for structural variant analysis. Short-read technologies generally produce sequence data that doesn’t span the variants, making it impossible to align and assemble them accurately. Long-read technology has shown great promise, but has been too expensive for large-scale, genome-wide analyses, the authors note.

So they developed a target-capture approach to enrich for structural variants at particular chromosomal locations. With oligo capture, they target specific insert sizes using the Pippin Prep for fragments up to 1 Kb and BluePippin for anything larger. After library prep is completed, the selected DNA is sequenced on a PacBio instrument. The process is known as the PacBio-LITS (large-insert targeted capture-sequencing) method and is especially noteworthy because it’s the first report of targeted sequencing for libraries with insert sizes greater than 1 Kb.

In this method, size selection is an essential step to the success of the pipeline. “Manual gel-extraction methods involving agarose gel electrophoresis can be used, but we have chosen Sage Science’s Pippin and BluePippin platforms to perform target size selection for improved accuracy and sample recovery,” the authors write, adding that they use “range mode” to preserve DNA complexity from the sample.

The Baylor team presents data from a study of three samples from patients with Potocki–Lupski syndrome. Scientists used PacBio-LITS to analyze structural rearrangements associated with the disease, looking particularly at breakpoint junctions of low-copy repeats (LCR). “We successfully identified previously determined breakpoint junctions … and also were able to discover novel junctions in repetitive sequences, including LCR-mediated breakpoints,” the authors write.

The team posits that beyond structural variation, this new method could also be useful for validating indels and phasing haplotypes.

Check out the full paper: “PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations.”

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