Enzyme digestion

DNA concentrations of all samples were adjusted to a final amount of 10 ng of DNA for metagenomic library preparation with the NEBNext Ultra II FS DNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA, USA). Library preparation was performed according to the manufacturer’s instructions with a few modifications. In brief, the DNA was fragmented using enzyme digestion (New England Biolabs, MA, USA) for 15 min and the Illumina adapters were diluted 25-fold to 0.6 µM to avoid the formation of primer dimers. Size selection was conducted with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), to select fragments of 250–400 bp length, purify libraries and eliminate residual primer-dimers (1:0.6 DNA to bead ratio). The indexing PCR was performed with 10 cycles. Library size and concentration were evaluated using a 5200 Fragment analyzer system (Agilent, Santa Clara, CA, USA) and a DNF-473 Standard Sensitivity NGS Fragment Analysis Kit (Agilent, CA, USA). Libraries were pooled equimolarly (final concentration of 4 nM), and 17 pM of the mixture was spiked with 1% PhiX and sequenced on NextSeq Illumina platform (Illumina Inc., San Diego, CA, USA) using the paired-end mode 2 × 150 bp Kit v 2.5 (Illumina Inc., CA, USA).

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).