Capturing diverse microbial sequence with comprehensive and scalable probe design

Abstract

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. We developed CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa, and implemented it in a publicly available software package. CATCH designs compact probe sets that achieve full coverage of known microbial sequence diversity and that scale well with this diversity. Using CATCH, we designed and synthesized multiple probe sets, including one to capture whole genomes of the 356 viral species known to infect humans, and conducted a rigorous evaluation of their performance. Capture with these probe sets enriched unique viral content on average 18× in sequencing libraries from patient and environmental samples and allowed us to assemble viral genomes that we could not otherwise recover. We show that capture accurately reflects co-infections and within-host nucleotide variation, enriches sequence with substantial divergence from the probe sets, and improves detection of viral infections in samples with unknown microbial content. Our work provides a new approach to probe design and evaluation, and demonstrates a path toward more sensitive, cost-effective metagenomic sequencing.

Enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples

“…Efficacy of probes varies by homology to the viral target, as evidenced by our results and those in the literature [13,15]. For example, the reference sequence used to design the probe set for DENV-1 exhibits 74% nucleotide identity over 35% of the length of the closest sequenced reference for the DENV-2 strain that was spiked.…”

“…While the probes employed in the studies published to date vary in length from 50-to 120-mers, enrichment methods also can differ by the number of probes and target viruses included in a set. An additional potential protocol difference is the number of samples pooled, which ranges from a single sample to 12 [9,10,15]. The current recommendation by Illumina for viral enrichment is to pool four samples [14].…”

“…The technical advancements of the RNA Access kit had already enabled the sequencing of previously unsequencable materials such as those of low concentration and formalin-fixed paraffin-embedded (FFPE) tissue [12], and now this protocol has been employed not only for the detection and characterization of EBOV from clinical samples but also for detection and characterization of respiratory viruses in clinical samples [13,14]. In general, hybridization-based viral target enrichment has been successfully employed to characterize viruses found within both contrived samples and clinical samples [3,9,10,13,[15][16][17][18][19][20]. The performance of the Respiratory Virus Panel (RVP) version of this method [14], which uses probes for 34 common respiratory viruses in conjunction with the TruSeq RNA Access protocol, was recently investigated and it was demonstrated to work well overall when tested on human clinical samples [13].…”

Co-circulating mumps lineages at multiple geographic scales

“…Hybrid capture Viral hybrid capture was performed as previously described 8 using two different probe sets. In one case, probes were created to target MuV and measles virus (V-MM probe set), and in one case, probes were created to target 356 species of viruses known to infect humans (V-All probe set) 9 . Capture using V-All was used to enrich viral sequences primarily in samples in which we could not detect MuV, as well as in other samples (see Supplementary Table 1 for a list of which samples were captured using which probe set).…”

“…We set out to investigate the evolution and spread of MuV, generating 203 whole genomes (160 collected in Massachusetts and 43 in 15 other states) through a combination of unbiased and capturebased sequencing approaches 8,9 (see Methods, Supplementary Table 1). The genomes had a median of 99.48% unambiguous base calls and a median depth of 176x (Extended Data Fig.…”