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 evaluate enrichment efficiency of V ALL , we prepared sequencing libraries from 30 patient and environmental samples containing at least one of 8 different viruses: dengue virus (DENV), GB virus C (GBV-C), Hepatitis C virus (HCV), HIV-1, Influenza A virus (IAV), Lassa virus (LASV), mumps virus (MuV), and Zika virus (ZIKV) (see Supplementary Table 2 for details). These 8 viruses together reflect a range of typical viral titers in biological samples, including ones that have extremely low levels, such as ZIKV 38, 39 . The samples encompass a range of source materials: plasma, serum, buccal swabs, urine, avian swabs, and mosquito pools. We performed capture on these libraries and sequenced them both before and after capture. To compare enrichment of viral content across sequencing runs, we downsampled raw read data from each sample to the same number of reads (200,000) before further analysis. Downsampling to correct for differences in sequencing depth, rather than the more common use of a normalized count such as reads per million, is useful for two reasons. First, it allows us to compare our ability to assemble genomes (e.g., owing to capture) in samples that were sequenced to different depths. Second, downsampling helps to correct for differences in sequencing depth in the presence of a high frequency of PCR duplicate reads (see Methods for details), as observed in captured libraries. We removed duplicate reads during analyses so that we could measure enrichment of viral information (i.e., unique viral content) rather than measure an artifactual enrichment arising from PCR amplification.