Truseq dna cd indexes

Two NGS libraries with 24 individuals each were prepared. Targeting an average fragment size of 350 bp, 500 ng of DNA was sheared by sonication using a Bioruptor Pico (Diagenode, Belgium) with seven cycles of 15 s ‘on’ and 90 s ‘off’ at 6 °C. Aiming for an even coverage across the length of the plastomes, library preparation was performed using the TruSeq DNA PCR-Free Low Throughput Library Prep Kit (Illumina, USA), including indexed adapters from the TruSeq DNA CD Indexes (Illumina) according to the manufacturer’s protocol. Individual libraries were pooled, targeting an equal representation of each individual in the final libraries. Both libraries were sequenced on an Illumina HiSeq 2500 at VBCF Vienna NGS Unit (http://vbcf.ac.at/ngs/) as 125 bp paired-end reads. All generated genomic data are deposited as a BioProject at the NCBI Sequence Read Archive (BioProject ID PRJNA419625, SRA Study ID SRP142704; SRA accessions for each sample are given in Supplementary Data Table S1).

Twenty-one samples with desired levels of quality subsequently underwent HTS library preparation using the TruSeq^® Stranded Total RNA Library Prep Plant (96 samples) and TruSeq DNA CD Indexes (96 indexes, 96 samples) (Illumina, San Dieago, CA, USA) by following the manufacturer’s instructions. All samples were pooled and sequenced using the Miseq-Illumina platform at CCOVI, Brock University, Canada. Approximately one million pair end reads on average were generated for each sample (average size ranging from 116.9 to 137.7 bp—Table 3).

A biotinylated DNA probe-based capture method was applied to sequence KIR and HLA genes to high-resolution, as described previously (33 (link)). Briefly, 500 ng genomic DNA for each sample was randomly fragmented using enzyme digestion (New England Biolabs, Boston, MA) and then labeled with TruSeq DNA CD indexes (Illumina Inc, San Diego, CA). Fragments of 800–1,200 bp length were obtained by size selection and the individual samples pooled at equal quantity into one tube. Fragments containing target HLA and KIR genes were captured using specific probes, prepared to a final concentration of 12 pmol/L, as determined by Qubit instrument, then sequenced using a MiSeq instrument and a v3 Reagent Kit (600-cycle; Illumina Inc, San Diego, CA, USA).