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

Despite the potential of capture, there are challenges and practical considerations that are present with the use of any probe set. Notably, as capture requires additional cycles of amplification, computational analyses should properly account for duplicate reads due to amplification; the inclusion of unique molecular identifiers 54, 55 could improve determination of unique fragments. Also, quantifying the sensitivity and specificity of capture with comprehensive probe sets is challenging -as it is for metagenomic sequencing more broadly -because doing so would necessitate obtaining viral genomes for the hundreds of targeted species, and false positives are likely to be due to components of sequencing and classification that are unrelated to capture (e.g., contamination in sample processing or read misclassifications). For sequencing some ultra low input samples, targeted amplicon approaches may be faster and more sensitive 38 , but genome size, sequence heterogeneity, and the need for prior knowledge of the target species can limit the feasibility and sensitivity of these approaches 1, 56, 57 . Similarly, for molecular diagnostics of particular pathogens, many commonly used assays such as qRT-PCR and rapid antigen tests are likely to be faster and less expensive than metagenomic sequencing. Capture does increase the preparation cost and time per-sample compared to unbiased metagenomic sequencing, but this is offset by reduced sequencing costs through increased sample pooling and/or lower-depth sequencing 1 (Supplementary Table 9 ).