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Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
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Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
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Journal Article
Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
2021
Overview
The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.
Publisher
Public Library of Science,Public Library of Science (PLoS)
Subject
/ COVID-19
/ DNA, Complementary - chemistry
/ DNA, Complementary - metabolism
/ Genomes
/ Genomics
/ Humans
/ Medicine
/ Medicine and health sciences
/ Open Reading Frames - genetics
/ Patients
/ Real-Time Polymerase Chain Reaction
/ Research and analysis methods
/ RNA
/ SARS-CoV-2 - isolation & purification
/ Sequence Analysis, DNA - methods
/ Severe acute respiratory syndrome
/ Severe acute respiratory syndrome coronavirus 2
/ Virology
/ Workflow
