Baaijens, J. A. | Search bioRxiv

bioRxiv2023

Tracking SARS-CoV-2 variants of concern in wastewater: an assessment of nine computational tools using simulated genomic data

Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic "novel" lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances, and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1% frequency, results were more reliable above a 5% threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of noise or bias in wastewater sequencing data and to appreciate the commonalities and differences across methods.

bioinformatics↗

bioRxiv2023

AmpliDiff: An Optimized Amplicon Sequencing Approach to Estimating Lineage Abundances in Viral Metagenomes

Metagenomic profiling algorithms commonly rely on genomic differences between lineages, strains, or species to infer the relative abundances of sequences present in a sample. This observation plays an important role in the analysis of diverse microbial communities, where targeted sequencing of 16S and 18S ribosomal RNA (rRNA), both well-known hypervariable genomic regions, have led to insights into microbial diversity and the discovery of novel organisms. However, the variable nature of discriminatory regions can also act as a double-edged sword, as the sought-after variability can make it difficult to design primers for their amplification through Polymerase Chain Reaction (PCR). Moreover, the most variable regions are not necessarily the most informative regions for the purpose of differentiation; one should focus on regions that maximize the number of lineages that can be distinguished. Here we present AmpliDiff, a computational tool that simultaneously finds such highly discriminatory genomic regions, as well as primers allowing for the amplification of these regions. We show that regions and primers found by AmpliDiff can be used to accurately estimate relative abundances of SARS-CoV-2 lineages, for example in wastewater sequencing data. We obtain mean absolute prediction errors that are comparable with using whole genome information to estimate relative abundances. Furthermore, our results show that AmpliDiff is robust against incomplete input data and that primers designed by AmpliDiff continue to bind to genomes originating from months after the primers were selected. With AmpliDiff we provide an effective and efficient alternative to whole genome sequencing for estimating lineage abundances in viral metagenomes.

bioinformatics↗