Capturing diverse microbial sequence with comprehensive and scalable probe design

Hayden C. Metsky; Katherine J. Siddle; Adrianne Gladden-Young; James Qu; David K. Yang; Patrick Brehio; Andrew Goldfarb; Anne Piantadosi; Shirlee Wohl; Amber Carter; Aaron E. Lin; Kayla G. Barnes; Damien C. Tully; Björn Corleis; Scott Hennigan; Giselle Barbosa-Lima; Yasmine R. Vieira; Lauren M. Paul; Amanda L. Tan; Kimberly F. Garcia; Leda A. Parham; Ikponmwonsa Odia; Philomena Eromon; Onikepe A. Folarin; Augustine Goba; Etienne Simon-Lorière; Lisa Hensley; Angel Balmaseda; Eva Harris; Douglas Kwon; Todd M. Allen; Jonathan A. Runstadler; Sandra Smole; Fernando A. Bozza; Thiago M. L. Souza; Sharon Isern; Scott F. Michael; Ivette Lorenzana; Lee Gehrke; Irene Bosch; Gregory Ebel; Donald Grant; Christian Happi; Daniel J. Park; Andreas Gnirke; Pardis C. Sabeti; Christian B. Matranga

We synthesized the 349,998 probes in V ALL using the SeqCap EZ Developer platform (Roche). Since the number of features on the array was 2.1 million, we repeated the design 6 times (6× final probe density). We used these biotinylated single-stranded DNA probes directly for hybrid capture experiments. We performed in solution hybridization and capture according to manufacturer instructions (SeqCapEZ v5.1) with modifications to make the protocol compatible with Nextera XT libraries. Specifically, we pooled up to 6 individual sequencing libraries with at least 1 unique index together at equimolar concentrations (≥ 3 nm) in a final volume of 50 µL. We replaced the manufacturer's indexed adapter blockers with oligos complementary to Nextera indexed adapters (P7 blocking oligo: 5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACN NNN NNN NTC GTC GGC AGC GTC AGA TGT GTA TAA GAG ACA G/3ddC/-3'; P5 blocking oligo: 5'-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN NNG TCT CGT GGG CTC GGA GAT GTG TAT AAG AGA CAG /3ddC/-3'; Integrated DNA Technologies). The concentration of Nextera XT adapter blockers was reduced to 200 µm to account for sample input < 1 µg. The concentration of probes was also reduced to account for the replication of our V ALL probe set 6× across the 2.1 million features. We incubated the hybridization reaction overnight (∼ 16 hrs). After hybridization and capture on streptavidin beads, we amplified library pools using PCR (14-16 cycles) with universal Illumina PCR primers (P7 primer: 5'-CAAGCAGAAGACGGCATACGA-3'; P5 primer: 5'-AATGATACGGCGACCACCGA-3'; Integrated DNA Technologies).