Sureselectxt target enrichment system

In this study, HLA regions were enriched with an in-solution bait capture and the SureSelectXT Target Enrichment System (Illumina) for the Illumina paired-end multiplexed sequencing library. Using a UDG-treated sequencing library and a custom bait library designed by Michael Wittig et al.^{15 (link)}, the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA, and HLA-DPB1) were enriched in 68 St. Jørgen samples. Samples were pooled (up to four) per capture with 800 ng of library DNA per pool in a volume of 3.4 µL. Hybridization buffer and blocking reagent were handled according to the manufacturer’s instructions. As the captured samples were indexed already, 1 µL of each IS5 and IS6 primers (100 µM) were used for indexing/amplification instead of the primers provided in the capture kit. The post-capture PCR cycle number was set to 12. Purification of the amplified captured libraries was performed according to the protocol using AMPure XP beads. The quality of the captured library pools was assessed on the Agilent 2100 Bioanalyzer with  the High Sensitivity DNA Assay. The sequencing was carried out on the Illumina HiSeq 4000 (2 × 75 cycles) platform at the Institute of Clinical Molecular Biology, Kiel University, using the HiSeq v4 chemistry and the manufacturer’s protocol for multiplex sequencing.

The library preparation for capturing of selected DNA regions was performed according to the SureSelect XT Target Enrichment System protocol for Illumina paired-end sequencing (Agilent). In brief, 3 μg of genomic DNA was sheared on a Covaris™ E220 focused-ultrasonicator. Fragment size (150-200 bp) and quantity were confirmed with an Agilent 2100 Bioanalyzer 7500 chip. The fragmented DNA was end-repaired, adenylated and ligated to Agilent indexing-specific paired-end adaptors. The DNA with adaptor-modified ends was PCR amplified (6 cycles, Herculase II fusion DNA polymerase) with SureSelect primers, quality controlled using the DNA 7500 assay specific for a library size of 250–350 bp, and hybridized for 24 hr at 65°C. The hybridization mixture was washed in the presence of magnetic beads (Dynabeads MyOne Streptavidin T1, Life Technologies), and the eluate PCR amplified (16 cycles) to add index tags using SureSelectXT Indexes for Illumina. The final library size and concentration was determined using an Agilent 2100 Bioanalyzer 7500 chip and sequenced on an Illumina HiSeq 2000 platform with a paired-end run of 2 × 76 bp, following the manufacturer’s protocol. 36 sample libraries were loaded in three lanes of HiSeq 2000.

Library preparation of the 52 family members of 12 families collected at the Radboudumc, Nijmegen was performed with the SureSelect^XT target enrichment system for Illumina paired‐end multiplex sequencing according to manufacturer's instructions (Version B4, August 2015, Agilent Technologies). Completed libraries were sent to the Department of Genetics of Maastricht University Medical Center+, Maastricht, the Netherlands, where sequencing was performed with eight samples per lane using an Illumina HiSeq2000 with 2*100 bp chemistry, together with a large cohort of 269 sporadic CSC patients (Schellevis et al., 2018 submitted).
The 20 samples of the six families collected at Leiden University Medical Center, Leiden, the Netherlands were sent to GenomeScan BV, Leiden, for sequencing. For these samples, the Agilent SureSelect V5 enrichment kit was used and sequencing was performed with 2*125 bp chemistry on the HiSeq2500.