Validation of Variant Assembly Using HAPHPIPE with Next-Generation Sequence Data from Viruses

Abstract

Next-generation sequencing (NGS) offers a powerful opportunity to identify low-abundance, intra-host viral sequence variants, yet the focus of many bioinformatic tools on consensus sequence construction has precluded a thorough analysis of intra-host diversity. To take full advantage of the resolution of NGS data, we developed HAplotype PHylodynamics PIPEline (HAPHPIPE), an open-source tool for the de novo and reference-based assembly of viral NGS data, with both consensus sequence assembly and a focus on the quantification of intra-host variation through haplotype reconstruction. We validate and compare the consensus sequence assembly methods of HAPHPIPE to those of two alternative software packages, HyDRA and Geneious, using simulated HIV and empirical HIV, HCV, and SARS-CoV-2 datasets. Our validation methods included read mapping, genetic distance, and genetic diversity metrics. In simulated NGS data, HAPHPIPE generated pol consensus sequences significantly closer to the true consensus sequence than those produced by HyDRA and Geneious and performed comparably to Geneious for HIV gp120 sequences. Furthermore, using empirical data from multiple viruses, we demonstrate that HAPHPIPE can analyze larger sequence datasets due to its greater computational speed. Therefore, we contend that HAPHPIPE provides a more user-friendly platform for users with and without bioinformatics experience to implement current best practices for viral NGS assembly than other currently available options.

HAPHPIPE: Haplotype Reconstruction and Phylodynamics for Deep Sequencing of Intrahost Viral Populations

“…To place our software in the context of existing bioinformatics tools, we discuss and compare several alternative viral assembly pipelines all with respect to HAPHPIPE in our validation study ( Gibson et al. 2020b ).…”

“…As most existing viral NGS platforms focus on HIV-1 exclusively, this represents a significant advantage of HAPHPIPE for users analyzing many different viral species. For a thorough comparison of HAPHPIPE’s methods with those of other tools, please see Gibson et al. (2020b) .…”

“…Often haplotypes are preferred for estimating transmission clusters ( Gibson et al. 2020a ), testing for associations between clinical variables (phenotypes) and genetic variation within individuals versus among individuals ( Gibson et al. 2020a ), and characterizing drug resistance variants ( Johnson et al.…”

“…HAPHPIPE has been thoroughly compared with similar viral assembly tools and has been validated against two commonly used programs: HyDRA and Geneious ( Gibson et al. 2020b ).…”

A large population sample of African HIV genomes from the 1980s reveals a reduction in subtype D over time associated with propensity for CXCR4 tropism

“…A visual assessment in Geneious Prime 2022.0.1 ( www.geneious.com ) was carried out to check for good coverage across the genome, or any dips that might indicate an inter-subtype recombinant sequence. If this was the case, the multi-reference BAM files were examined, or an alternative de-novo assembly with HAPHPIPE and SPAdes was attempted [ 5 , 29 ]. Either the single reference assembly (or de-novo assembly if improvement could be found) was then fed into the HAPHPIPE framework for fine tuning with three rounds of iterative improvement.…”