TNM
DNA was purified using AMPure XP (Beckman Coulter Life Sciences, Brea, CA, USA), quantified using Qubit DNA HS Assay Kit (Thermo Fisher, Waltham, MA, USA), and normalized to 20 ng/L. 200 ng of DNA were digested by MspI (4 u) and PstI-HF (4 u) restriction enzymes. After enzyme inactivation at 70 C, 18 L of 2 Instant Sticky-end Ligase Master Mix (New England Biolabs, Ipswich, MA, USA), and 1 L each of 25 nM double-stranded P5-GBS-PstI and P7-GBS-MspI adapters with modified bases were added to the mix. After 1 h ligation at 22 C, DNA was sedimented using magnetic KAPA Beads (Roche, Switzerland) and sticky ends were blunted by BST polymerase. Long fragments and concatenates were removed using AMPureXP. Indexing PCR was performed with 6 nmoles of the universal S4_indPCR primer and 6 nmoles of the corresponding indexing primer with Phusion HotStart PCR MasterMix (NEB, Ipswich, MA, USA) with the following profile: 30 s at 98 C; 15 cycles of 10 s at 98 C, 30 s at 60 C, and 5 s at 72 C; final extension step for 10 min at 72 C. Libraries prepared for different specimens were pooled in equimolar quantities; the 240600 bp fraction was excised by BluePippin (Sage Sciences, Beverly, MA, USA). Sequencing was performed using 150 cycles NextSeq500/550 High Output Kit v2.5 (Illumina, San Diego, CA, USA), the Read1_GBS_PstI, and the custom indexing primer [19].