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

The segments of the H4N4 genome display different levels of enrichment following capture ( Supplementary Fig. 9 ). To investigate whether these differences are related to sequence divergence from the probes, we compared the identity between probes and sequence in the H4N4 genome to the observed enrichment of that sequence (Fig. 3b) . We saw the least enrichment in segment 6 (N), which had the least identity between probe sequence and the H4N4 sequence, as we did not include any sequences of the N4 subtypes in the probe designs. Interestingly, V ALL did show limited positive enrichment of segment 6, as well as of segment 4 (H); these enrichments were lower than those of the less divergent segments. But this was not the case for segment 4 when using V WAFR , suggesting a greater target affinity of V WAFR capture when there is some degree of divergence between probes and target sequence ( Fig. 3b) , potentially due to this probe set's longer, ssRNA probes. For both probe sets, we observed no clear inter-segment differences in enrichment across the remaining segments, whose sequences have high identity with probe sequences (Fig. 3b, Supplementary Fig. 9 ). These results show that the probe sets can capture sequence that differs markedly from what they were designed to target, but nonetheless that sequence similarity with probes influences enrichment efficiency.