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

To analyze the relation between probe-target identity and enrichment (Fig. 3b) , we used an Influenza A virus sample of avian subtype H4N4 (IAV-SM5). We assembled a genome of this sample both pre-capture and following capture with V ALL to verify concordance; we used the V ALL sequence for further analysis here because it was more complete. We aligned depleted reads to this genome as described above (with BWA using the align and plot coverage function of viral-ngs and the following parameters: -m 50000 --excludeDuplicates --aligner options '-k 12 -B 2 -O 3' --minScoreToFilter 60). For a window in the genome, we calculated the fold-change in depth to be the fold-change of the mean depth post-capture against the mean depth pre-capture within the window. Here, we used windows of length 150 nt, sliding with a stride of 25 nt. We aligned all probe sequences in V ALL and V WAFR designs to this genome using BWA-MEM 86 with the following options: -a -M -k 8 -A 1 -B 1 -O 2 -E 1 -L 2 -T 20; these sensitive parameters should account for most possible hybridizations, and include a low soft-clipping penalty to allow us to model a portion of a probe hybridizing to a target while the remainder hangs off. We counted the number of bases that match between a probe and target sequence using each alignment's MD tag (this does not count soft-clipped ends), and defined the identity between a probe and target sequence to be this number of matching bases divided by the probe length. We defined the identity between probes and a window of the target genome as follows: we considered all mapped probe sequences that have at least half their alignment within the window, and took the mean of the top 25% of identity values between these probes and the target sequence. In Fig. 3b , we plot a point for each window. We did this separately with probes from the V ALL and V WAFR designs.