VirPool: Model-Based Estimation of SARS-CoV-2 Variant Proportions in Wastewater Samples (preprint)

Background: The genomes of SARS-CoV-2 are classified into variants, some of which are monitored as variants of concern (e.g. the delta variant B.1.617.2 or omicron variant B.1.1.529). Proportions of these variants in a population are typically estimated by large-scale sequencing of individual patient samples. Sequencing a mixture of SARS-CoV-2 RNA molecules from wastewater provides a cost-effective alternative, but requires methods for estimating variant proportions in a mixed sample. Results: We propose a new method based on a probabilistic model of sequencing reads, capturing sequence diversity present within individual variants, as well as sequencing errors. The algorithm is implemented in an open source Python program called VirPool. We evaluated the accuracy of VirPool on several simulated and real sequencing data sets from both Illumina and nanopore sequencing platforms, including wastewater samples from Austria and France monitoring the onset of alpha and delta variants. Conclusions: VirPool is a versatile tool for wastewater and other mixed-sample analysis that can handle both short- and long-read sequencing data. Our approach does not require pre-selection of characteristic mutations for variant profiles, it is able to use the entire length of reads instead of just the most informative positions, and can also capture haplotype dependencies within a single read.

Sequencing SARS-CoV-2 in Slovakia: An Unofficial Genomic Surveillance Report (preprint)

We present an unofficial SARS-CoV-2 genomic surveillance report from Slovakia based on approximately 3500 samples sequenced between March 2020 and May 2021. Early samples show multiple independent imports of SARS-CoV-2 from other countries. In Fall 2020, three virus variants (B.1.160, B.1.1.170, B.1.258) dominated as the number of cases increased. In November 2020, B.1.1.7 (alpha) variant was introduced in Slovakia and quickly became the most prevalent variant in the country (>75% of new cases by early February 2021 and >95% in mid-March).

Technical comment on The impact of population-wide rapid antigen testing on SARS-CoV-2 prevalence in Slovakia (preprint)

Pavelka et al. (Science, Reports, 7 May 2021) claim that a single round of population-wide antigen testing in Slovakia reduced the observed COVID-19 prevalence by 58%, and that it played a substantial role in curbing the pandemic. We argue that this estimate, which is based on incorrect assumptions, is exaggerated, and that the relief was short-lived with little effect on mitigating the pandemic.

Nanopore Sequencing of SARS-CoV-2: Comparison of Short and Long PCR-tiling Amplicon Protocols (preprint)

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with this approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, as well as the reads derived from the viral sub-genomic RNAs and/or human and bacterial contamination.

A novel, multiplexed RT-qPCR assay to distinguish lineage B.1.1.7 from the remaining SARS-CoV-2 lineages (preprint)

The emergence of a novel SARS-CoV-2 variant called lineage B.1.1.7 sparked global alarm due to evidence of increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect lineage B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack robust genomic surveillance programs and failed assays detect multiple unrelated variants containing similar mutations as B.1.1.7, we sought to develop an RT-qPCR test that can accurately and rapidly differentiate the B.1.1.7 variant from other SARS-CoV-2 variants. We used bioinformatics, allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop a test that differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing. Our room temperature-stable, multiplexed RT-qPCR test consists of two assays that target either the common SARS-CoV-2 spike gene or spike gene deletions specific to lineage B.1.1.7. A simple relative comparison of the Ct values of the two assays permits not only identification of the B.1.1.7 variant but also its differentiation from other variants that harbor only the {Delta}H69/{Delta}V70 deletion. The test showed 97% clinical sensitivity at detecting lineage B.1.1.7. This test can easily be implemented in labs to rapidly scale B.1.1.7 surveillance efforts and is particularly useful in countries with high prevalence of variants possessing only the {Delta}H69/{Delta}V70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Time-adjusted Analysis Shows Weak Associations Between BCG Vaccination Policy and COVID-19 Disease Progression (preprint)

In this study, we ascertain the associations between BCG vaccination policies and progression of COVID-19 through analysis of various time-adjusted indicators either directly extracted from the incidence and death reports, or estimated as parameters of disease progression models. We observe weak correlation between BCG vaccination status and indicators related to disease reproduction characteristics. We did not find any associations with case fatality rates (CFR), but the differences in CFR estimates are at present likely dominated by differences in testing and case reporting between countries.