tetano
Editor, Senior Moderator
PLoS One
. 2021 Aug 25;16(8):e0244468.
doi: 10.1371/journal.pone.0244468. eCollection 2021.
Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
Harsha Doddapaneni[SUP] 1 [/SUP], Sara Javornik Cregeen[SUP] 2 3 [/SUP], Richard Sucgang[SUP] 2 [/SUP], Qingchang Meng[SUP] 1 [/SUP], Xiang Qin[SUP] 1 [/SUP], Vasanthi Avadhanula[SUP] 3 [/SUP], Hsu Chao[SUP] 1 [/SUP], Vipin Menon[SUP] 1 [/SUP], Erin Nicholson[SUP] 3 4 [/SUP], David Henke[SUP] 3 [/SUP], Felipe-Andres Piedra[SUP] 3 [/SUP], Anubama Rajan[SUP] 3 [/SUP], Zeineen Momin[SUP] 1 [/SUP], Kavya Kottapalli[SUP] 1 [/SUP], Kristi L Hoffman[SUP] 2 3 [/SUP], Fritz J Sedlazeck[SUP] 1 [/SUP], Ginger Metcalf[SUP] 1 [/SUP], Pedro A Piedra[SUP] 3 4 [/SUP], Donna M Muzny[SUP] 1 [/SUP], Joseph F Petrosino[SUP] 2 3 [/SUP], Richard A Gibbs[SUP] 1 [/SUP]
Affiliations
Abstract
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.
. 2021 Aug 25;16(8):e0244468.
doi: 10.1371/journal.pone.0244468. eCollection 2021.
Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
Harsha Doddapaneni[SUP] 1 [/SUP], Sara Javornik Cregeen[SUP] 2 3 [/SUP], Richard Sucgang[SUP] 2 [/SUP], Qingchang Meng[SUP] 1 [/SUP], Xiang Qin[SUP] 1 [/SUP], Vasanthi Avadhanula[SUP] 3 [/SUP], Hsu Chao[SUP] 1 [/SUP], Vipin Menon[SUP] 1 [/SUP], Erin Nicholson[SUP] 3 4 [/SUP], David Henke[SUP] 3 [/SUP], Felipe-Andres Piedra[SUP] 3 [/SUP], Anubama Rajan[SUP] 3 [/SUP], Zeineen Momin[SUP] 1 [/SUP], Kavya Kottapalli[SUP] 1 [/SUP], Kristi L Hoffman[SUP] 2 3 [/SUP], Fritz J Sedlazeck[SUP] 1 [/SUP], Ginger Metcalf[SUP] 1 [/SUP], Pedro A Piedra[SUP] 3 4 [/SUP], Donna M Muzny[SUP] 1 [/SUP], Joseph F Petrosino[SUP] 2 3 [/SUP], Richard A Gibbs[SUP] 1 [/SUP]
Affiliations
- PMID: 34432798
- DOI: 10.1371/journal.pone.0244468
Abstract
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.