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

Abstract

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.

A two-pronged approach for rapid and high-throughput SARS-CoV-2 nucleic acid testing

“…The maximum volume of NP swab-VTM specimen that could be added to the m-RT-PCR was 5.5 µl (e.g., 10.99 µl of VTM/QE mixture). [7][8][9][10][11][12][13][14][15] and cross contamination between samples is a major concern. To minimize contamination, we followed procedures established for working in clinical molecular laboratories including separation of pre-PCR and post-PCR laboratory space, the use of a dedicated hood for making dilutions, sterilizing Eppendorf pipettes with 10% bleach followed by UV-light treatment for 30 minutes.…”

“…Scalability of this workflow during this COVID-19 pandemic has been hampered by supply shortages in NP swabs and RT-qPCR assay reagents, and by limited sample throughput capabilities and extended time to report test results. Recently, several innovative approaches for SARS-CoV-2 testing have been developed such as analysis of human saliva specimens with [3][4][5][6][7] or without a prior RNA purification step 8 , a rapid colorimetric assay 9 or a CRISPR-based assay 10 using reversetranscription loop-mediated isothermal amplification (RT-LAMP), an amplification-free CRISPR-Cas13a-based mobile phone assay 11 , and SARS-CoV-2 detection using next-generation sequencing as a readout [12][13][14][15] . Successful implementation of these approaches into workable testing solutions could dramatically increase SARS-CoV-2 diagnostic capacity and throughput while reducing the time to receive test results.…”

Role of Multiomics Data to Understand Host–Pathogen Interactions in COVID-19 Pathogenesis

“… 53 NGS-based approaches have allowed for the detection of more than 2–3 viral genomes or transcriptome fragments that can be detected easily using methods such as SARSeq (saliva analysis by RNA sequencing), which can detect the presence of SARS-CoV-2 and other respiratory viruses such as influenza A and B. 54 Moreover, the metagenomic NGS (mNGS) protocol that allows detection of SARS-CoV-2 and other associated pathogens present in a clinical sample at once has aided the discovery of coinfections that would have been otherwise missed. 55 In a similar study, a higher abundance of Propionibacteriaceae and reductions in Corynebacterium accolens was observed in COVID-19 negative patients compared to COVID-19 positive patients.…”

“…For example, targeted analysis of structural spike S glycoprotein and replicase polyprotein-1ab peptides, QIAPGQTGK and AIVSTIQRKYK, respectively, are being explored. 126 MS methodology was further modified to build a paper spray MS for fast and accurate diagnosis within 60 s. 127 The very recently developed technique SARSeq, an NGS-based testing method, that can perform analysis of up to 36,000 samples in a single run 54 can resolve the problem of mass testing of COVID-19 at the time of a massive outbreak. Another recently developed in-house metagenomic NGS (mNGS) can detect SARS Cov2 and associated all other pathogens in a clinical sample, which eliminates the necessity of using multiple test kits to detect comorbidity.…”

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

“…To circumvent these limitations, several alternative technologies have been developed 9 . These are based on the detection of antigens [10][11][12][13] , colorimetric assays [14][15][16][17][18] , CRISPR-based detection [19][20][21][22] or multiplexed detection of nucleic acids using NGS [23][24][25][26][27][28][29] .…”