Improved detection of artifactual viral minority variants in high-throughput sequencing data

textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs)

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020: silent circulation

BACKGROUND: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. METHODS: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. RESULTS: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. CONCLUSION: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks

Distinct fragmentation patterns of circulating viral cell-free DNA in 83,552 non-invasive prenatal testing samples

Aim: The fragmentation characteristics of cell-free DNA (cfDNA) are informative biomarkers in liquid biopsies, including non-invasive prenatal testing (NIPT), as they provide insights into the origins of the cfDNA. Viral infections by DNA viruses can contribute to the available cfDNA in these samples. Here, we characterize the fragment size distribution of viral cfDNA fragments obtained from available anonymous NIPT samples.Methods: A viral database of 224 DNA viruses was generated from the NCBI RefSeq viral database. Paired-end cfDNA sequencing reads from 83.522 NIPT samples that did not map to any of the human chromosomes, or mitochondrial DNA of the human reference genome build GRCh38 (excluding alternative and unplaced contigs) were remapped to the generated viral database. Reads mapping to the 14 most abundant DNA viruses were selected, and fragment size distributions were analyzed in detail.Results: Distinct fragmentation patterns were identified for several DNA viruses, most likely due to differences in viral tropism, chromatinization (binding of nucleosomes), and the topology of the viral DNA. In high viral load parvo B19 positive samples, the fragment size distribution differed between samples, potentially reflecting the state of the infection.Conclusion: These findings outline the potential for liquid biopsies to elucidate the dynamics behind the viral infection, which may potentially have various clinical applications. Our data provide preliminary insights on the use of fragmentomics of viral cfDNA to distinguish between reactivation, reinfection, and primary infection and monitoring the state of viral infections

Clinically relevant DNA viruses in pregnancy

Infections by DNA viruses during pregnancy are associated with increased health risks to both mother and fetus. Although not all DNA viruses are related to an increased risk of complications during pregnancy, several can directly infect the fetus and/or cause placental dysfunction. During Non-Invasive Prenatal Testing analysis, the presence of viral DNA can be detected, theoretically allowing screening early in pregnancy. Although treatment options are currently limited, this might rapidly change in the near future. It is therefore important to be aware of the potential impact of these viruses on feto-maternal health. In this manuscript we provide a brief introduction into the most commonly detected DNA viruses in human cell-free DNA sequencing experiments and their pathogenic potential during pregnancy

The cell-free DNA virome of 108,349 Dutch pregnant women

Objective: Viral infections during pregnancy are a major health concern to mother and fetus. By repurposing cell-free Non Invasive Prenatal Testing (NIPT) sequencing data, we investigated prevalence and abundance of viral DNA in a cohort of 108,349 pregnant women. Method: Cell-free DNA (cfDNA) sequencing reads that did not map to any of the human chromosomes or mitochondrial DNA of the human reference genome build GRCh38 were aligned to 224 DNA viruses selected from the NCBI refseq viral database. Results: In total 443,665 reads of viral origin were detected across 42,273 samples representing 165 viral species. Several are known to be potentially harmful during pregnancy and/or childbirth, including Cytomegalovirus, Parvovirus B19 and Hepatitis B. Viral sequences were mostly detected at very low abundance. However, several cases had exceptionally high viral loads for Parvovirus B19, Hepatitis B and others. We found statistically significant associations between presence of viral DNA and gestational age, maternal age, fetal fraction, cfDNA concentration and others. Conclusion: We demonstrate the feasibility to detect viral DNA from typical genome-wide NIPT cfDNA sequencing and describe the main characteristics of the viral DNA in our cohort. Our dataset of detected viral sequence reads is made publicly available to guide future clinical implementations

Possible host-adaptation of SARS-CoV-2 due to improved ACE2 receptor binding in mink.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on mink farms are increasingly observed in several countries, leading to the massive culling of animals on affected farms. Recent studies showed multiple (anthropo)zoonotic transmission events between humans and mink on these farms. Mink-derived SARS-CoV-2 sequences from The Netherlands and Denmark contain multiple substitutions in the S protein receptor binding domain (RBD). Molecular modeling showed that these substitutions increase the mean binding energy, suggestive of potential adaptation of the SARS-CoV-2 S protein to the mink angiotensin-converting enzyme 2 (ACE2) receptor. These substitutions could possibly also impact human ACE2 binding affinity as well as humoral immune responses directed to the RBD region of the SARS-CoV-2 S protein in humans. We wish to highlight these observations to raise awareness and urge for the continued surveillance of mink (and other animal)-related infections

Deep sequencing identifies hepatitis B virus core protein signatures in chronic hepatitis B patients

Background: We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients. Methods: Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg-positive and -negative patients, and between patients with combined response and non-response to peginterferon/adefovir combination therapy. Results: We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg-positive and -negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non-response. The fraction non-consensus sequence on selected positions was significantly higher in HBeAg-negative patients, and was negatively correlated with HBV DNA and HBsAg levels. Conclusions: Sequence Harmony identified a number of amino acid changes associated with HBeAg-status and response to peginterferon/adefovir combination therapy

Development of Resistance-Associated Mutations after Sotrovimab Administration in High-risk Individuals Infected with the SARS-CoV-2 Omicron Variant

This study investigates whether resistance-associated mutations develop after treatment with sotrovimab in high-risk patients infected with the SARS-CoV-2 Omicron variant

Viral population analysis and minority-variant detection using short read next-generation sequencing

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitr