scholarly journals Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients

2020 ◽  
Author(s):  
Yanqun Wang ◽  
Daxi Wang ◽  
Lu Zhang ◽  
Wanying Sun ◽  
Zhaoyong Zhang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Evolutionary Dynamics ◽  
Single Patient ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Host Interactions ◽  
Genomic Variants ◽  
Deep Sequencing Data ◽  
Serial Samples

ABSTRACTAs of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

scholarly journals Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yanqun Wang ◽  
Daxi Wang ◽  
Lu Zhang ◽  
Wanying Sun ◽  
Zhaoyong Zhang ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Respiratory Tract ◽  
High Throughput Sequencing ◽  
Evolutionary Dynamics ◽  
Rapid Development ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Host Interactions ◽  
Single Nucleotide Variations ◽  
Serial Samples

Abstract Background Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Methods Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). Results The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. Conclusions Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.

scholarly journals Identification of single nucleotide variants using position-specific error estimation in deep sequencing data

2018 ◽  
Author(s):  
Dimitrios Kleftogiannis ◽  
Marco Punta ◽  
Anuradha Jayaram ◽  
Shahneen Sandhu ◽  
Stephen Q. Wong ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Low Frequency ◽  
Poisson Model ◽  
Real Data ◽  
Analytical Sensitivity ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Deep Sequencing Data ◽  
Targeted Deep Sequencing

AbstractBackgroundTargeted deep sequencing is a highly effective technology to identify known and novel single nucleotide variants (SNVs) with many applications in translational medicine, disease monitoring and cancer profiling. However, identification of SNVs using deep sequencing data is a challenging computational problem as different sequencing artifacts limit the analytical sensitivity of SNV detection, especially at low variant allele frequencies (VAFs).MethodsTo address the problem of relatively high noise levels in amplicon-based deep sequencing data (e.g. with the Ion AmpliSeq technology) in the context of SNV calling, we have developed a new bioinformatics tool called AmpliSolve. AmpliSolve uses a set of normal samples to model position-specific, strand-specific and nucleotide-specific background artifacts (noise), and deploys a Poisson model-based statistical framework for SNV detection.ResultsOur tests on both synthetic and real data indicate that AmpliSolve achieves a good trade-off between precision and sensitivity, even at VAF below 5% and as low as 1%. We further validate AmpliSolve by applying it to the detection of SNVs in 96 circulating tumor DNA samples at three clinically relevant genomic positions and compare the results to digital droplet PCR experiments.ConclusionsAmpliSolve is a new tool for in-silico estimation of background noise and for detection of low frequency SNVs in targeted deep sequencing data. Although AmpliSolve has been specifically designed for and tested on amplicon-based libraries sequenced with the Ion Torrent platform it can, in principle, be applied to other sequencing platforms as well. AmpliSolve is freely available at https://github.com/dkleftogi/AmpliSolve.

scholarly journals Identification of single nucleotide variants using position-specific error estimation in deep sequencing data

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Dimitrios Kleftogiannis ◽  
Marco Punta ◽  
Anuradha Jayaram ◽  
Shahneen Sandhu ◽  
Stephen Q. Wong ◽  
...  
Keyword(s):  
Error Estimation ◽  
Deep Sequencing ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Deep Sequencing Data ◽  
Specific Error

scholarly journals SARS-CoV-2 genomic analyses in cancer patients reveal elevated intrahost genetic diversity

2021 ◽  
Author(s):  
Juliana D Siqueira ◽  
Livia R Goes ◽  
Brunna M Alves ◽  
Pedro S de Carvalho ◽  
Claudia Cicala ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
Cancer Patients ◽  
Disease Severity ◽  
Healthcare Workers ◽  
Viral Population ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Consensus Sequences ◽  
Vulnerable Patients

Abstract Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (non-cancer controls) by deep sequencing and investigated the within-host viral population of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers from the Brazilian National Cancer Institute were collected in April–May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%) among the consensus sequences. Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to healthcare workers. This difference was independent of SARS-CoV-2 Ct values obtained at the diagnostic tests, which did not differ between the two groups. The most common nucleotide changes of intrahost SNVs in both groups were consistent with APOBEC and ADAR activities. Intrahost genetic diversity in cancer patients was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Moreover, the presence of metastasis, either in general or specifically in the lung, was not associated with intrahost diversity among cancer patients. Cancer patients carried significantly higher numbers of minor variants compared to non-cancer counterparts. Further studies on SARS-CoV-2 diversity in especially vulnerable patients will shed light onto the understanding of the basis of COVID-19 different outcomes in humans.

scholarly journals Implications of Genetic Distance to Reference and De Novo Genome Assembly for Clinical Genomics in Africans

2020 ◽  
Author(s):  
Daniel Shriner ◽  
Adebowale Adeyemo ◽  
Charles Rotimi
Keyword(s):  
Genetic Distance ◽  
De Novo ◽  
Reference Sequence ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
De Novo Genome Assembly ◽  
Single Nucleotide ◽  
Clinical Genomics ◽  
Advantages And Disadvantages ◽  
False Discovery

In clinical genomics, variant calling from short-read sequencing data typically relies on a pan-genomic, universal human reference sequence. A major limitation of this approach is that the number of reads that incorrectly map or fail to map increase as the reads diverge from the reference sequence. In the context of genome sequencing of genetically diverse Africans, we investigate the advantages and disadvantages of using a de novo assembly of the read data as the reference sequence in single sample calling. Conditional on sufficient read depth, the alignment-based and assembly-based approaches yielded comparable sensitivity and false discovery rates for single nucleotide variants when benchmarked against a gold standard call set. The alignment-based approach yielded coverage of an additional 270.8 Mb over which sensitivity was lower and the false discovery rate was higher. Although both approaches detected and missed clinically relevant variants, the assembly-based approach identified more such variants than the alignment-based approach. Of particular relevance to individuals of African descent, the assembly-based approach identified four heterozygous genotypes containing the sickle allele whereas the alignment-based approach identified no occurrences of the sickle allele. Variant annotation using dbSNP and gnomAD identified systematic biases in these databases due to underrepresentation of Africans. Using the counts of homozygous alternate genotypes from the alignment-based approach as a measure of genetic distance to the reference sequence GRCh38.p12, we found that the numbers of misassemblies, total variant sites, potentially novel single nucleotide variants (SNVs), and certain variant classes (e.g., splice acceptor variants, stop loss variants, missense variants, synonymous variants, and variants absent from gnomAD) were significantly correlated with genetic distance. In contrast, genomic coverage and other variant classes (e.g., ClinVar pathogenic or likely pathogenic variants, start loss variants, stop gain variants, splice donor variants, incomplete terminal codons, variants with CADD score ≥20) were not correlated with genetic distance. With improvement in coverage, the assembly-based approach can offer a viable alternative to the alignment-based approach, with the advantage that it can obviate the need to generate diverse human reference sequences or collections of alternate scaffolds.

scholarly journals Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew Currin ◽  
Neil Swainston ◽  
Mark S Dunstan ◽  
Adrian J Jervis ◽  
Paul Mulherin ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequencing ◽  
Cost Effective ◽  
Polymorphism Analysis ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Design Build ◽  
Hardware Costs

Abstract Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

scholarly journals neoepiscope improves neoepitope prediction with multivariant phasing

2019 ◽  
Vol 36 (3) ◽  
pp. 713-720 ◽  
Author(s):  
Mary A Wood ◽  
Austin Nguyen ◽  
Adam J Struck ◽  
Kyle Ellrott ◽  
Abhinav Nellore ◽  
...  
Keyword(s):  
False Negative ◽  
Supplementary Information ◽  
Supplementary File ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Somatic Variant ◽  
Negative Results ◽  
Multiple Datasets ◽  
False Negative Results

Abstract Motivation The vast majority of tools for neoepitope prediction from DNA sequencing of complementary tumor and normal patient samples do not consider germline context or the potential for the co-occurrence of two or more somatic variants on the same mRNA transcript. Without consideration of these phenomena, existing approaches are likely to produce both false-positive and false-negative results, resulting in an inaccurate and incomplete picture of the cancer neoepitope landscape. We developed neoepiscope chiefly to address this issue for single nucleotide variants (SNVs) and insertions/deletions (indels). Results Herein, we illustrate how germline and somatic variant phasing affects neoepitope prediction across multiple datasets. We estimate that up to ∼5% of neoepitopes arising from SNVs and indels may require variant phasing for their accurate assessment. neoepiscope is performant, flexible and supports several major histocompatibility complex binding affinity prediction tools. Availability and implementation neoepiscope is available on GitHub at https://github.com/pdxgx/neoepiscope under the MIT license. Scripts for reproducing results described in the text are available at https://github.com/pdxgx/neoepiscope-paper under the MIT license. Additional data from this study, including summaries of variant phasing incidence and benchmarking wallclock times, are available in Supplementary Files 1, 2 and 3. Supplementary File 1 contains Supplementary Table 1, Supplementary Figures 1 and 2, and descriptions of Supplementary Tables 2–8. Supplementary File 2 contains Supplementary Tables 2–6 and 8. Supplementary File 3 contains Supplementary Table 7. Raw sequencing data used for the analyses in this manuscript are available from the Sequence Read Archive under accessions PRJNA278450, PRJNA312948, PRJNA307199, PRJNA343789, PRJNA357321, PRJNA293912, PRJNA369259, PRJNA305077, PRJNA306070, PRJNA82745 and PRJNA324705; from the European Genome-phenome Archive under accessions EGAD00001004352 and EGAD00001002731; and by direct request to the authors. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Clonal propagation history shapes the intra-cultivar genetic diversity in ‘Malbec’ grapevines

2020 ◽  
Author(s):  
Luciano Calderón ◽  
Nuria Mauri ◽  
Claudio Muñoz ◽  
Pablo Carbonell-Bejerano ◽  
Laura Bree ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Clonal Propagation ◽  
Variant Calling ◽  
Red Wines ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Diversity Pattern ◽  
History Of ◽  
Potential Impact ◽  
Whole Genome Resequencing

AbstractGrapevine (Vitis vinifera L.) cultivars are clonally propagated to preserve their varietal attributes. However, novel genetic variation still accumulates due to somatic mutations. Aiming to study the potential impact of clonal propagation history on grapevines intra-cultivar genetic diversity, we have focused on ‘Malbec’. This cultivar is appreciated for red wines elaboration, it was originated in Southwestern France and introduced into Argentina during the 1850s. Here, we generated whole-genome resequencing data for four ‘Malbec’ clones with different historical backgrounds. A stringent variant calling procedure was established to identify reliable clonal polymorphisms, additionally corroborated by Sanger sequencing. This analysis retrieved 941 single nucleotide variants (SNVs), occurring among the analyzed clones. Based on a set of validated SNVs, a genotyping experiment was custom-designed to survey ‘Malbec’ genetic diversity. We successfully genotyped 214 samples and identified 14 different clonal genotypes, that clustered into two genetically divergent groups. Group-Ar was driven by clones with a long history of clonal propagation in Argentina, while Group-Fr was driven by clones that have longer remained in Europe. Findings show the ability of such approaches for clonal genotypes identification in grapevines. In particular, we provide evidence on how human actions may have shaped ‘Malbec’ extant genetic diversity pattern.

scholarly journals Misannotation of multiple-nucleotide variants risks misdiagnosis

2019 ◽  
Vol 4 ◽  
pp. 145
Author(s):  
Matthew N. Wakeling ◽  
Thomas W. Laver ◽  
Kevin Colclough ◽  
Andrew Parish ◽  
Sian Ellard ◽  
...  
Keyword(s):  
Best Practices ◽  
False Negative ◽  
Simulated Data ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Public Resources ◽  
Next Generation Sequencing Analysis ◽  
Optimal Approach

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses that would previously have been made by standard Sanger (dideoxy) sequencing. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. This risk will be increased as confirmatory Sanger sequencing of Single Nucleotide variants (SNVs) ceases to be standard practice. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

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