Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats

Abstract

Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel alpha- and betacoronaviruses in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.

Molecular detection using hybridization capture and next-generation sequencing reveals cross-species transmission of feline coronavirus type-1 between a domestic cat and a captive wild felid

“…This is especially true when targeting the S gene of Alphacoronavirus-1, which does not always yield reliable results (60). In contrast, it has been shown that by using HC, it is possible to detect and sequence complete genomes of multiple CoVs in a single sample, as well as to discover new CoVs (61).…”

“…This is especially true when targeting the S gene of Alphacoronavirus-1 , which does not always yield reliable results (60). In contrast, it has been shown that by using HC, it is possible to detect and sequence complete genomes of multiple CoVs in a single sample, as well as to discover new CoVs (61). Implementing the hybridization capture method reported in this study to characterize different Alphacoronavirus -1 and to identify co-infections will be essential to determine if wild felid species could act as mixing vessels for the emergence of recombinant genotypes of FCoV or Alphacoronavirus-1 in general.…”

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology

“…Out of Asia, Kuchinski et al [ 222 ] designed baits against all bat CoV sequences (18,365 baits) for recovery of CoVs from bat swabs in the Democratic Republic of the Congo. An additional 1635 baits were designed from conserved motifs in non-bat CoVs to round the number of baits up to 20,000.…”

“…Long reads are suitable for accurate assemblies, which have downstream analytical implications such as comparative genomics and pathogen identification [ 273 , 274 ]. In a response letter to reviewers by Kuchinski et al [ 222 ], they found several limitations of MinION sequencing in conjunction with HC. Most importantly, they only managed to recover sequences comparable in length to those from Illumina libraries, defeating the purpose of using long-read sequencers.…”

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using synthetic viral sequences and clinical samples

“…For example, depletion of host cells prior to extraction of nucleic acids (NA) has been reported to be not advantageous in clinical samples as also intracellular viral particles or NA will be removed 11,12 . In contrast, viral enrichment by probe hybridization methods has been reported to significantly increase sensitivity in various sample types 1,[13][14][15][16][17][18][19] , up to the level required for accurate detection of low frequency virus variants 20 .…”