Library preparation for Illumina sequencing

For the paired-end sequencing, the DNA was fragmented to roughly 300 bp using a Covaris M220 focused-ultrasonicator (Covaris, Woburn, Massachusetts, United States). The library was then prepared using the KAPA LTP Library Preparation Kit (KAPABiosystems, Wilmington, Massachusetts, United States) and quantified using the KAPA Library Quantification Kit (KAPABiosystems), following the manufacturer’s instructions. The paired-end library was sequenced on a NextSeq500 (Illumina) using one Mid and one High Output v2 kit.

Two mate-pair libraries, with intended insert sizes of 4,500 bp and 7,000 bp, were prepared using the Nextera Mate-Pair Library Preparation kit (Illumina), following the manufacturer’s instructions. The mate-pair libraries were quantified using the KAPA Library Quantification Kit (KAPABiosystems) and sequenced on a NextSeq500 (Illumina). However, when later investigated bioinformatically, both libraries seemed to have an insert size of approximately 2 kbp. This was most likely because of error in the library preparation and/or fragmented DNA. One of the libraries was sequenced with a High Output v2 kit while the other was sequenced with a Mid Output v2 kit.