Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing

Bloom, Joshua S.; Sathe, Laila; Munugala, Chetan; Jones, Eric M.; Gasperini, Molly; Lubock, Nathan B.; Yarza, Fauna; Thompson, Erin; Kovary, Kyle M.; Park, Jimin; Marquette, Dawn; Kay, Stephania; Lucas, Mark; Love, TreQuan; Booeshaghi, A. Sina; Brandenberg, Oliver F.; Guo, Longhua; Boocock, James; Hochman, Myles; Simpkins, Scott W.; Lin, Isabella; LaPierre, Nathan; Hong, Duke; Zhang, Yi; Oland, Gabriel; Choe, Bianca Judy; Chandrasekaran, Sukantha; Hilt, Evann E.; Butte, Manish J.; Damoiseaux, Robert; Kravit, Clifford; Cooper, Aaron; Yin, Yi; Pachter, Lior; Garner, Omai B.; Flint, Jonathan; Eskin, Eleazar; Luo, Chongyuan; Kosuri, Sriram; Kruglyak, Leonid; Arboleda, Valerie A.

doi:10.1101/2020.08.04.20167874

Cited by 49 publications

(82 citation statements)

References 25 publications

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“…Swab-Seq, another method that applies next generation sequencing to the diagnosis of SARS-COV-2, employs unique molecular barcodes embedded in RT-PCR primers to enable parallel sequencing of thousands of samples using very short reads in a single run [ 89 ].…”

Section: Next Generation Sequencingmentioning

confidence: 99%

Molecular diagnostic assays for COVID-19: an overview

Habibzadeh

Mofatteh

Silawi

et al. 2021

Critical Reviews in Clinical Laboratory Sciences

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The coronavirus disease 2019 (COVID-19) pandemic has highlighted the cardinal importance of rapid and accurate diagnostic assays. Since the early days of the outbreak, researchers with different scientific backgrounds across the globe have tried to fulfill the urgent need for such assays, with many assays having been approved and with others still undergoing clinical validation. Molecular diagnostic assays are a major group of tests used to diagnose COVID-19. Currently, the detection of SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR) is the most widely used method. Other diagnostic molecular methods, including CRISPR-based assays, isothermal nucleic acid amplification methods, digital PCR, microarray assays, and next generation sequencing (NGS), are promising alternatives. In this review, we summarize the technical and clinical applications of the different COVID-19 molecular diagnostic assays and suggest directions for the implementation of such technologies in future infectious disease outbreaks.

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Section: Next Generation Sequencingmentioning

confidence: 99%

Molecular diagnostic assays for COVID-19: an overview

Habibzadeh

Mofatteh

Silawi

et al. 2021

Critical Reviews in Clinical Laboratory Sciences

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“…V-seq has a number of potential applications beyond SARS-CoV-2 genome sequencing in positive patient samples. Several NGS-based approaches for high-throughput SARS-CoV-2 testing have recently been developed (12). Briefly, these methods use a small number of viral-specific RT primers with sequencing adaptors, followed by detection based on normalized viral read counts.…”

Section: Discussionmentioning

confidence: 99%

Rapid cost-effective viral genome sequencing by V-seq

Guo

Boocock

Tome

et al. 2020

Preprint

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Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3 prime end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.

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“…Analyzing an extended cohort using an optimized multiplex panel v2.0. SARS-CoV-2 RNA concentration spans a large dynamic range, such that spike-in mutant amplicons which have been suggested to improve performance of NGS-based strategies 16 might interfere with detection of COVID-19-positive cases with low viral reads. Therefore, we instead used our NSA data to create multiplex panel v2.0 (see the "Methods" section) that removed primers yielding NSAs by targeting a distinct region of RdRP,…”

Section: Development Of a C19-spar-seq Diagnostic Platform To Detectmentioning

confidence: 99%

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2

et al. 2021

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Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

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Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing

Cited by 49 publications

References 25 publications

Molecular diagnostic assays for COVID-19: an overview

Molecular diagnostic assays for COVID-19: an overview

Rapid cost-effective viral genome sequencing by V-seq

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2

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