ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa

Abstract

Background Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico. In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools’ suitability for challenging viral probe design applications. Results Based on ProbeTool’s in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools’ in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results. Conclusions ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools’ design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.

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

“…All available bat CoV sequences were downloaded from NCBI GenBank on 4 October 2020. A custom panel of 20,000 hybridization probes was designed from these sequences using the ProbeTools package (v0.0.5) ( Kuchinski et al, 2022c ). All available sequences in the following taxa were downloaded from NCBI GenBank on 4 October 2020: unclassified coronavirinae (txid: 693995), unclassified coronaviridae (txid: 1986197), alphacoronavirus (txid: 693996), and betacoronavirus (txid: 694002).…”

“…Bat CoV sequences were extracted by searching sequence headers for bat-related key words identified by the authors. These sequences were used as targets for probe design with the ProbeTools package (v0.0.5) ( https://github.com/KevinKuchinski/ProbeTools ; copy archived at swh:1:rev:20f78c3af2e88be28ac6130b3588f5c16e49c7a6 ; Kuchinski et al, 2022c ; Kuchinski, 2022b ). All possible probes were generated from the bat CoV sequences using the makeprobes module with a batch size of 100 probes.…”

Targeted genomic sequencing of avian influenza viruses in wetlands sediment from wild bird habitats

“…material from swine barns and agricultural fairs, filtered air from building HVAC systems, and municipal wastewater) [39][40][41][42][43] . Additionally, these specimens often contain other pathogens of importance to agriculture and public health, and probe panels could be expanded for simultaneous multi-pathogen detection [44][45][46][47] . In this way, the probe capture-based targeted genomic sequencing platform demonstrated here could provide a powerful general-purpose tool for pathogen surveillance.…”

“…First, all reference sequences representing a segment were clustered at 99% nucleotide identity using VSEARCH cluster_fast (v2.21.0) without masking (qmask none) 50 . Cluster centroids were used as the input design space for ProbeTools makeprobes (v0.1.9) using batch sizes of 10 probes (-b 10), probe length of 120 nucleotides (-k 120), and a coverage endpoint of 95% (-c 95) 44 . Sub-panels for each IAV genome segment were combined to create the final panel.…”

High performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

“…50 ProbeTools (v.0.1.9) modules capture and stats were used to align the pan-HAV probe sequences against the selected target sequences to assess probe coverage and depth. 51 ProbeTools getlowcov was used to find low coverage regions, defined as 120-kmers with zero probe coverage at more than 40 consecutive sites. A Mann-Whitney U test was performed to evaluate whether the low probe coverage regions identified in the control sequence had significantly lower read depth compared to the rest of the genome in enriched samples using scipy (v.1.18.1).…”