Journal Article

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing

García-Sastre, Adolfo, Miorin, Lisa, Cohen, Phillip, Patel, Roosheel S, DeGrace, Emma J, Barrall, Erika A, Danziger, Oded, Kehrer, Thomas, Bobrowski, Tesia, Rosenberg, Brad R, Cupic, Anastasija

2023

Overview

Single cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNA-Seq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended read 1 (R1) sequencing strategy maximizes the detection of sgmRNAs by increasing the number of unambiguous reads spanning leader-sgmRNA junction sites. Using this method, we show that viral gene expression is highly correlated across cells suggesting a relatively consistent proportion of viral sgmRNA production throughout infection. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Coronaviruses / COVID-19 / Gene expression / Genomics / gRNA / Microbiology / Severe acute respiratory syndrome coronavirus 2 / Transcription