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 first removed contaminating DNA by treatment with TURBO DNase (Ambion) and prepared double-stranded cDNA by priming with random hexamers followed by synthesis of the second strand as previously described 14 . We used the Nextera XT kit (Illumina) to prepare sequencing libraries with modifications to enable hybrid capture 10 . Specifically, we used non-biotinylated i5 indexing primers (Integrated DNA Technologies) in place of the manufacturer's standard i5 PCR primers. As cDNA concentrations from clinical samples are typically lower than the recommended 1 ng, input to Nextera XT was 5 µL of cDNA, except in the case of Ebola serial dilutions where input was 1 ng. Samples underwent 16-18 cycles of PCR and final libraries were quantified using either the 2100 Bioanalyzer dsDNA High Sensitivity assay (Agilent) or by qPCR using the KAPA Universal Complete Kit (Roche). We also prepared sequencing libraries from water with each batch as a negative control.