Rapid cost-effective viral genome sequencing by V-seq

Guo, Longhua; Boocock, James; Tome, Jacob M.; Chandrasekaran, Sukantha; Hilt, Evann E.; Zhang, Yi; Sathe, Laila; Li, Xinmin; Luo, Chongyuan; Kosuri, Sriram; Shendure, Jay; Arboleda, Valerie A.; Flint, Jonathan; Eskin, Eleazar; Garner, Omai B.; Yang, Shangxin; Bloom, Joshua S.; Kruglyak, Leonid; Yin, Yi

doi:10.1101/2020.08.15.252510

Cited by 13 publications

(17 citation statements)

References 12 publications

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“…The next aspect we evaluated was when to add the DNA indices that distinguish individual samples. These can in principle be incorporated during the RT 15,18,19 as well as during the PCR 24,25 , as extensions of the primers used to reverse transcribe or amplify the desired amplicons, respectively (Fig. 1C) .…”

Section: Methods Development and Resultsmentioning

confidence: 99%

SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

Yelagandula

Bykov

Vogt

et al. 2020

Preprint

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During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massive parallel, yet cost effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.

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Section: Methods Development and Resultsmentioning

confidence: 99%

SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

Yelagandula

Bykov

Vogt

et al. 2020

Preprint

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“…We recently developed V-seq, a sequencing method that uses virus-specific RT primers tiled across the SARS-CoV-2 genome for viral sequence enrichment (Guo et al, 2020). The V-seq protocol is more rapid and 10 times cheaper than commercially available meta-transcriptomics approaches ( e.g ., NEBNext Ultra II).…”

Section: Resultsmentioning

confidence: 99%

“…For reads assigned to primer set oP2, we combined the 8 base-pair UMI with the 6 base-pair random hexamer to create a 14 base-pair UMI. For a more detailed description of the primer design see (Guo et al, 2020). We used the GroupReadsByUmi tool from the fgbio (v1.2.0; https://github.com/fulcrum-genomics/fgbio) toolkit to group reads using this UMI.…”

Section: Methodsmentioning

confidence: 99%

“…We recently developed a rapid and inexpensive sequencing method based on targeted reverse transcription of the SARS-CoV-2 genome directly from patient RNA (Guo et al, 2020). We used this method, together with a meta-transcriptomic approach, to generate 142 high-quality viral genome sequences from patients residing in LA County who were seen at UCLA Health.…”

Section: Introductionmentioning

confidence: 99%

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Genomic epidemiology of the Los Angeles COVID-19 outbreak

Guo

Boocock

Hilt

et al. 2020

Preprint

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Los Angeles (LA) County has sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history of SARS-CoV-2 in LA County, we sequenced 142 viral genomes from unique patients seeking care at UCLA Health System. 86 of these genomes are from samples collected before April 19, 2020. We found that the early outbreak in LA, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a US-specific strain, B.1.43, which has been found predominantly in California and Washington State. While samples from LA County carry the ancestral B.1.43 genome, viral genomes from neighbouring counties in California and from counties in Washington State carry additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but may have been undermined by the many introductions of SARS-CoV-2 into the region. Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the U.S. to have coordinated inter-state responses to the pandemic.

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“…Reads mapping to viral sequences in samples containing no SARS-CoV-2 RNA are a source of false positive test outcomes and limit sensitivity of detection assays. Non-zero background has been observed in other sequencing-based assays for SARS-CoV-2 detection 23,24,53 , and molecular contamination and mis-assignment of sequencing reads were identified as main underlying sources 23 . Leveraging the UMI information we have shown that between 1% and 6% of the UMI counts in a pool are contaminants of another pool (Figure S7B), resulting from events taking place after reverse transcription (during library amplification or sequencing).…”

Section: Sources Of and Strategies To Improve Incomplete Umimentioning

confidence: 99%

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

Vonesch

Bredikhin

Dobrev

et al. 2020

Preprint

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McQ is a SARS-CoV-2 quantification assay that couples early-stage barcoding with high-throughput sequencing to enable multiplexed processing of thousands of samples. McQ is based on homemade enzymes to enable low-cost testing of large sample pools, circumventing supply chain shortages.Implementation of cost-efficient high-throughput methods for detection of RNA viruses such as SARS-CoV-2 is a potent strategy to curb ongoing and future pandemics. Here we describe Multiplexed SARS-CoV-2 Quantification platform (McQ), an in-expensive scalable framework for SARS-CoV-2 quantification in saliva samples. McQ is based on the parallel sequencing of barcoded amplicons generated from SARS- CoV-2 genomic RNA. McQ uses indexed, target-specific reverse transcription (RT) to generate barcoded cDNA for amplifying viral- and human-specific regions. The barcoding system enables early sample pooling to reduce hands-on time and makes the ap-proach scalable to thousands of samples per sequencing run. Robust and accurate quantification of viral load is achieved by measuring the abundance of Unique Molecular Identifiers (UMIs) introduced during reverse transcription. The use of homemade reverse transcriptase and polymerase enzymes and non-proprietary buffers reduces RNA to library reagent costs to 92 cents/sample and circumvents potential supply chain short-ages. We demonstrate the ability of McQ to robustly quantify various levels of viral RNA in 838 clinical samples and accu-rately diagnose positive and negative control samples in a test-ing workflow entailing self-sampling and automated RNA ex-traction from saliva. The implementation of McQ is modular, scalable and could be extended to other pathogenic targets in future.

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Rapid cost-effective viral genome sequencing by V-seq

Cited by 13 publications

References 12 publications

SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

Genomic epidemiology of the Los Angeles COVID-19 outbreak

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

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