Amplicon-based targeted sequencing of EBV BALF2: human plasma
We designed 28 conserved primers to generate 14 small overlapping amplicons for targeted enrichment. Given the small insert size (median 153 bases, range 93-219), fragmentation was not performed and adapter sequences were included at the 5 end of each primer. Each of the 28 primers was separately synthesized with read 1 and read 2 adapters. Even- and odd-numbered primersets were pooled separately. Extracted nucleic acid was amplified by PCR in two reactions using 12.5 L LongAmp Taq 2X Mastermix Hot Start (New England BioLabs, Ipswich, MA), 0.3125 L of 2 M read 1 odd or even-numbered primersets (25 nM each primer), 0.3125 L of 2 M read 2 odd or even-numbered primersets (25 nM each primer), 1.875 L nuclease-free water, and 10.0 L template. Target enrichment PCR conditions were 94C for 0:30, and then 45 cycles of 94C for 00:30, 57.0C for 00:30, and 65C for 01:00 prior to final 65C extension for 10:00.  Libraries were prepared using NEBNext library preparation reagents for Illumina sequencing instruments (New England BioLabs, Ipswich, MA). Half of the products from the two PCR reactions (25 L total) were pooled and purified with 45 L (1.8X ratio) AMPure XP beads (Beckman Coulter, Brea, CA) and 80% ethanol, then resuspended into 25 L AVE elution buffer. Libraries were then indexed using dual index primers in a 50 L reaction containing 25 L NEBNext UltraTM II Q5 Master Mix (M0544), 1 L index 1 (i7) primer, 1 L index 2 (i5) primer, and 23 L amplified DNA. Thermocycler settings for indexing were: 98C for 00:30, then 12 cycles of 98C for 00:10 and 65.0C for 01:15 prior to final extension at 65.0C for 05:00.  Indexed libraries were then purified with 90 L (1.8X ratio) AMPure XP beads and 80% ethanol, then resuspended into 40 L AVE elution buffer. Individual indexed specimens were then pooled and normalized (1-20uL each) based on EBNA-1 qPCR viral load. The pooled library was then purified with a 1:1 ratio of AMPure XP beads and 80% ethanol, then resuspended into 40 L AVE elution buffer. Pooled indexed library fragment size was measured with a BioAnalyzer 2100. The library was then quantified using the Qubit dsDNA broad range assay and diluted to 15 pM. Each sequencing run contained one no template control, one wild-type B95-8 control, and up to 94 clinical specimens. Libraries were sequenced on an Illumina MiSeq using 150-cycle sequencing using the MiSeq Nano reagent kit V2.