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Scientists from the Wellcome Trust Sanger Institute recently published “An improved approach to mate-paired library preparation for Illumina sequencing,” a paper that came out in the open access journal Methods in Next Generation Sequencing.

The publication, from lead author Naomi Park, is the culmination of a project designed to establish an alternative to currently available commercial kits for mate-pair library construction. These existing methods of circularization “have differing limitations and are often linked to a single sequencing platform in a kit format, which may not be cost effective,” the authors write.

Mate-pair sequencing is often used for de novo genome assembly, detection of structural variants, genome finishing, and other projects in which long-range sequence data is beneficial. But preparing these libraries tends to be challenging — and gets even worse when there’s not much DNA to start with or when the DNA is degraded, the scientists note.

The paper compares a new method developed by the Sanger scientists with several existing mate-pair library construction approaches. “In order to generate unbiased and diverse Illumina mate-paired libraries containing even genomic sequence either side of a common adapter sequence, we altered the Illumina mate-pair protocol to use a modified SOLiD 4 hybridisation and ligation circularisation approach,” they write. Tests involving multiple libraries and sample types demonstrated that the new method increases library complexity and quality while reducing chimeras.

Most of the libraries involved in this study were size selected with BluePippin. “Alternative methods of size selection are amenable to this method, but may result in a differing breadth of size range and/or recovery,” the authors write. After comparing results from Pippin, manual gels, and bead size selection, they conclude, “The Blue Pippin provides the greatest recovery and accuracy of currently available commercial methods.”