Genomic library preparation
DNA extractions for supernatant, formalin-fixed and pellet samples were
sent directly to Arbor Biosciences for library preparation, capture, and
sequencing. Samples arrived at Arbor Biosciences fully dried and were
resuspended in 50 μl of Buffer EBT. Fragment distributions of a subset
of the gDNA samples were checked via Bioanalyzer (Agilent, Santa Clara,
CA). Samples were quantified via an intercalating dye assay, and gDNA
samples were sonicated and size-selected to a fragment length
< 300 bp. Up to 200 ng of sonicated gDNA, where available, was
taken into Illumina TruSeq-style library preparation. Samples received
5–7 cycles of indexing amplification to add unique dual-indexes.
Capture pools of five samples were prepared with 150 ng of gDNA
libraries. Captures were performed at 62°C (to account for potential
divergence of hDNA samples from bait sequence) overnight according to
the myBaits v4 manual. After clean-up, capture pools were amplified for
12 cycles followed by a second round of capture with 3x the recommended
amount of Block C (human cot-1; following McCartnery-Melstad et al.
2016) with the rest of the parameters remaining the same. The final
post-capture material was amplified for 10 cycles and sequenced on a
partial lane of a NovaSeq 6000 S4 PE150 run, resulting in 113.6 million
PE reads (~3.8 million reads per library).