SARS-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples

SARS-CoV-2 genomic surveillance has been vital in understanding the spread of COVID-19, the emergence of viral escape mutants and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low frequency variants. Our analyses highlight that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina respiratory viral oligo panel, and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high quality sequence data from low viral load samples for public health and genomic surveillance purposes.

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Sars-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.01046-21 ◽

2021 ◽

Author(s):

C. Lam ◽

K. Gray ◽

M. Gall ◽

R. Sadsad ◽

A. Arnott ◽

...

Keyword(s):

Viral Load ◽

Genome Sequencing ◽

Sequence Data ◽

Phylogenetic Analyses ◽

Low Frequency ◽

Variant Calling ◽

High Sensitivity ◽

Viral Escape ◽

Viral Loads ◽

Sensitivity Specificity

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520. Longitudinal Analysis of SARS-CoV-2 Viruses in Hospitalized Adults

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.714 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S325-S326

Author(s):

Lacy Simons ◽

Ramon Lorenzo-Redondo ◽

Hannah Nam ◽

Scott C Roberts ◽

Michael G Ison ◽

...

Keyword(s):

Viral Load ◽

Genome Sequencing ◽

Viral Genome ◽

Temporal Dynamics ◽

Population Level ◽

Hospital Staff ◽

Therapeutic Interventions ◽

Viral Loads ◽

Qrt Pcr ◽

Over Time

Abstract Background The rapid spread of SARS-CoV-2, the causative agent of Coronavirus disease 2019 (COVID-19), has been accompanied by the emergence of viral mutations, some of which may have distinct virological and clinical consequences. While whole genome sequencing efforts have worked to map this viral diversity at the population level, little is known about how SARS-CoV-2 may diversify within a host over time. This is particularly important for understanding the emergence of viral resistance to therapeutic interventions and immune pressure. The goal of this study was to assess the change in viral load and viral genome sequence within patients over time and determine if these changes correlate with clinical and/or demographic parameters. Methods Hospitalized patients admitted to Northwestern Memorial Hospital with a positive SARS-CoV-2 test were enrolled in a longitudinal study for the serial collection of nasopharyngeal specimens. Swabs were administered to patients by hospital staff every 4 ± 1 days for up to 32 days or until the patients were discharged. RNA was extracted from each specimen and viral loads were calculated by quantitative reverse transcriptase PCR (qRT-PCR). Specimens with qRT-PCR cycle threshold values less than or equal to 30 were subject to whole viral genome sequencing by reverse transcription, multiplex PCR, and deep sequencing. Variant populations sizes were estimated and subject to phylogenetic analysis relative to publicly available SARS-CoV-2 sequences. Sequence and viral load data were subsequently correlated to available demographic and clinical data. Results 60 patients were enrolled from March 26th to June 20th, 2020. We observed an overall decrease in nasopharyngeal viral load over time across all patients. However, the temporal dynamics of viral load differed on a patient-by-patient basis. Several mutations were also observed to have emerged within patients over time. Distribution of SARS-CoV-2 viral loads in serially collected nasopharyngeal swabs in hospitalized adults as determined by qRT-PCR. Samples were collected every 4 ± 1 days (T#1–8) and viral load is displayed by log(copy number). Conclusion These data indicate that SARS-CoV-2 viral loads in the nasopharynx decrease over time and that the virus can accumulate mutations during replication within individual patients. Future studies will examine if some of these mutations may provide fitness advantages in the presence of therapeutic and/or immune selective pressures. Disclosures Michael G. Ison, MD MS, AlloVir (Consultant)

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Quantitative SARS-CoV-2 viral-load curves in paired saliva and nasal swabs inform appropriate respiratory sampling site and analytical test sensitivity required for earliest viral detection

Journal of Clinical Microbiology ◽

10.1128/jcm.01785-21 ◽

2021 ◽

Author(s):

Emily S. Savela ◽

Alexander Winnett ◽

Anna E. Romano ◽

Michael K. Porter ◽

Natasha Shelby ◽

...

Keyword(s):

Viral Load ◽

Los Angeles ◽

Nasal Swab ◽

Sampling Site ◽

High Sensitivity ◽

Analytical Sensitivity ◽

Viral Loads ◽

Analytical Test ◽

Nasal Swabs ◽

High Analytical Sensitivity

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic transmission, curb the spread of variants, and maximize treatment efficacy. Low-analytical-sensitivity nasal-swab testing is commonly used for surveillance and symptomatic testing, but the ability of these tests to detect the earliest stages of infection has not been established. In this study, conducted between September 2020 and June 2021 in the greater Los Angeles County, California area, initially-SARS-CoV-2-negative household contacts of individuals diagnosed with COVID-19 prospectively self-collected paired anterior-nares nasal-swab and saliva samples twice daily for viral-load quantification by high-sensitivity RT-qPCR and digital-RT-PCR assays. We captured viral-load profiles from the incidence of infection for seven individuals and compared diagnostic sensitivities between respiratory sites. Among unvaccinated persons, testing saliva with a high-analytical-sensitivity assay detected infection up to 4.5 days before viral loads in nasal swabs reached concentrations detectable by low-analytical-sensitivity nasal-swab tests. For most participants, nasal swabs reached higher peak viral loads than saliva, but were undetectable or at lower loads during the first few days of infection. High-analytical-sensitivity saliva testing was most reliable for earliest detection. Our study illustrates the value of acquiring early (within hours after a negative high-sensitivity test) viral-load profiles to guide the appropriate analytical sensitivity and respiratory site for detecting earliest infections. Such data are challenging to acquire but critical to design optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics.

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Clinical utility of cycle threshold values in the context of COVID-19

10.21203/rs.3.rs-41867/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sonia N. Rao ◽

Davide Manissero ◽

Victoria Steele ◽

Josep Pareja

Keyword(s):

Viral Load ◽

Troponin I ◽

Severe Disease ◽

Clinical Information ◽

High Sensitivity ◽

Clinical Value ◽

Cycle Threshold ◽

Viral Loads ◽

Ct Value ◽

Viral Culture

Abstract BackgroundThe ability to predict likely prognosis and infectiousness for patients with COVID-19 would aid patient management decisions. Diagnosis is usually via real-time PCR and it is unclear whether the semi-quantitative capability of this method, determining viral load through cycle threshold (Ct) values, can be leveraged.ObjectivesWe aim to review available knowledge on correlations between SARS-COV-2 Ct values and patient- or healthcare-related outcomes to determine whether Ct values provide useful clinical information.SourcesA PubMed search was conducted on 1st June 2020 based on a search strategy of (Ct value OR viral load) AND SARS-CoV-2. Data was extracted from studies reporting on the presence or absence of an association between Ct values, or viral loads determined via Ct value, and clinical outcomes.ContentData from 18 studies were relevant for inclusion. One study reported on the correlation between Ct values and mortality and one study reported on the correlation between Ct values and progression to severe disease; both reported a significant association (p < 0.001 and p = 0.008, respectively). Fourteen studies reported on the correlation between Ct value or viral loads determined via Ct value and disease severity and an association was observed in 8 (57%) studies. Studies reporting on the correlation of viral load with biochemical and haematological markers showed an association with at least one marker, including increased lactate dehydrogenase (n = 4), decreased lymphocytes (n = 3) and increased high-sensitivity troponin I (n = 2). Two studies reporting on the correlation with infectivity showed that lower Ct values were associated with higher viral culture positivity.ImplicationsData suggest that lower Ct values may be associated with worse outcomes, and that Ct values may be useful in predicting the clinical course and prognosis of patients with COVID-19; however, further studies are warranted to confirm clinical value.

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Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

10.1101/2020.05.28.122648 ◽

2020 ◽

Cited By ~ 2

Author(s):

Nikki E. Freed ◽

Markéta Vlková ◽

Muhammad B. Faisal ◽

Olin K. Silander

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sequence Data ◽

Variant Calling ◽

Whole Genome ◽

Oxford Nanopore ◽

Cost Efficient ◽

Primer Sets ◽

Library Preparation Method

AbstractRapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

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Comprehensive fundamental somatic variant calling and quality management strategies for human cancer genomes

Briefings in Bioinformatics ◽

10.1093/bib/bbaa083 ◽

2020 ◽

Author(s):

Xiaoyu He ◽

Shanyu Chen ◽

Ruilin Li ◽

Xinyin Han ◽

Zhipeng He ◽

...

Keyword(s):

Genome Sequencing ◽

High Throughput Sequencing ◽

Cancer Genomics ◽

Sequence Data ◽

Human Cancer ◽

Management Strategies ◽

Variant Calling ◽

Cancer Genome ◽

Sequencing Data ◽

Cancer Genome Sequencing

Abstract Next-generation sequencing (NGS) technology has revolutionised human cancer research, particularly via detection of genomic variants with its ultra-high-throughput sequencing and increasing affordability. However, the inundation of rich cancer genomics data has resulted in significant challenges in its exploration and translation into biological insights. One of the difficulties in cancer genome sequencing is software selection. Currently, multiple tools are widely used to process NGS data in four stages: raw sequence data pre-processing and quality control (QC), sequence alignment, variant calling and annotation and visualisation. However, the differences between these NGS tools, including their installation, merits, drawbacks and application, have not been fully appreciated. Therefore, a systematic review of the functionality and performance of NGS tools is required to provide cancer researchers with guidance on software and strategy selection. Another challenge is the multidimensional QC of sequencing data because QC can not only report varied sequence data characteristics but also reveal deviations in diverse features and is essential for a meaningful and successful study. However, monitoring of QC metrics in specific steps including alignment and variant calling is neglected in certain pipelines such as the ‘Best Practices Workflows’ in GATK. In this review, we investigated the most widely used software for the fundamental analysis and QC of cancer genome sequencing data and provided instructions for selecting the most appropriate software and pipelines to ensure precise and efficient conclusions. We further discussed the prospects and new research directions for cancer genomics.

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Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Biology Methods and Protocols ◽

10.1093/biomethods/bpaa014 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Nikki E Freed ◽

Markéta Vlková ◽

Muhammad B Faisal ◽

Olin K Silander

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sequence Data ◽

Variant Calling ◽

Whole Genome ◽

Oxford Nanopore ◽

Cost Efficient ◽

Primer Sets ◽

Library Preparation Method

Abstract Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

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SARS-CoV-2 is detectable using sensitive RNA saliva testing days before viral load reaches detection range of low-sensitivity nasal swab tests

10.1101/2021.04.02.21254771 ◽

2021 ◽

Author(s):

Emily S. Savela ◽

Alexander Winnett ◽

Anna E. Romano ◽

Michael K. Porter ◽

Natasha Shelby ◽

...

Keyword(s):

Viral Load ◽

Early Detection ◽

Nasal Swab ◽

Limit Of Detection ◽

High Sensitivity ◽

Viral Loads ◽

Test Sensitivity ◽

Sensitivity Tests ◽

Nasal Swabs ◽

Low Sensitivity

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic spread of COVID-19, curb the spread of viral variants by travelers, and maximize efficacy of therapeutic treatments. We designed a study to evaluate the preferred test sensitivity and sample type (saliva and nasal swab) for detecting early infections of COVID-19. We performed a case-ascertained study to monitor household contacts of individuals recently diagnosed with a SARS-CoV-2 infection. From those individuals, we obtained twice-daily self-collected anterior-nares nasal swabs and saliva samples and quantified SARS-CoV-2 RNA viral loads in those samples using high-sensitivity RT-qPCR and RT-ddPCR assays. We found that SARS-CoV-2 RNA first appears in saliva and then in nasal-swab samples. A high-sensitivity (limit of detection of ~103 copies/mL) RNA test detected SARS-CoV-2 virus in saliva 1.5 to 4.5 days before the viral load in the paired nasal-swab samples exceeded the limit of detection of low-sensitivity tests. It was possible to observe a high (>107-108 copies/mL) viral load in saliva samples while the paired nasal swab was either negative or had low (~103 copies/mL) viral load. Our results indicate that both sampling site and test sensitivity must be considered to ensure early detection of SARS-CoV-2 infection: high-sensitivity tests that use saliva can detect SARS-CoV-2 infection days earlier than low-sensitivity tests that use nasal swabs. Furthermore, early in the infection, low-sensitivity tests that use nasal swabs may miss SARS-CoV-2-positive individuals with very high and potentially infectious viral loads in saliva.

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Moss enables high sensitivity single-nucleotide variant calling from multiple bulk DNA tumor samples

Nature Communications ◽

10.1038/s41467-021-22466-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chuanyi Zhang ◽

Mohammed El-Kebir ◽

Idoia Ochoa

Keyword(s):

Cancer Genomics ◽

Low Frequency ◽

Variant Calling ◽

High Sensitivity ◽

Single Sample ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

Additional Time ◽

Single Nucleotide ◽

Multiple Samples

AbstractIntra-tumor heterogeneity renders the identification of somatic single-nucleotide variants (SNVs) a challenging problem. In particular, low-frequency SNVs are hard to distinguish from sequencing artifacts. While the increasing availability of multi-sample tumor DNA sequencing data holds the potential for more accurate variant calling, there is a lack of high-sensitivity multi-sample SNV callers that utilize these data. Here we report Moss, a method to identify low-frequency SNVs that recur in multiple sequencing samples from the same tumor. Moss provides any existing single-sample SNV caller the ability to support multiple samples with little additional time overhead. We demonstrate that Moss improves recall while maintaining high precision in a simulated dataset. On multi-sample hepatocellular carcinoma, acute myeloid leukemia and colorectal cancer datasets, Moss identifies new low-frequency variants that meet manual review criteria and are consistent with the tumor’s mutational signature profile. In addition, Moss detects the presence of variants in more samples of the same tumor than reported by the single-sample caller. Moss’ improved sensitivity in SNV calling will enable more detailed downstream analyses in cancer genomics.

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RelocaTE2: a high resolution transposable element polymorphism mapping tool for population resequencing

10.7287/peerj.preprints.2447v1 ◽

2016 ◽

Cited By ~ 1

Author(s):

Jinfeng Chen ◽

Travis Wrightsman ◽

Susan R Wessler ◽

Jason E. Stajich

Keyword(s):

Transposable Element ◽

Sensitivity And Specificity ◽

Genome Sequencing ◽

Population Genetic ◽

Sequence Data ◽

False Positive Rate ◽

Rice Chromosome ◽

High Sensitivity ◽

Real Sequence ◽

Robust Solution

Background Transposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE transposition events or polymorphisms can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools. Methods We have developed the tool RelocaTE2 ( http://github.com/stajichlab/RelocaTE2 ) for identification of TE polymorphisms at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision. Results and Discussion The performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrates a higher level of sensitivity and specificity when compared to other tools. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE polymorphisms and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing.

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