Viral RNA was extracted from 140 uL of heat inactivated (30 minutes at 95°C, as part of protocol detailed in 24 (link)) saliva samples using the QIAamp viral RNA mini kit (QIAGEN). 100ng of viral RNA was used to generate cDNA using the SuperScript IV first strand synthesis kit (Invitrogen). Viral cDNA was then used to generate sequencing libraries using the Swift SNAP Amplicon SARS CoV2 kit with additional coverage panel and unique dual indexing (Swift Biosciences) which were sequenced on an Illumina Novaseq SP lane. Data were run through the nf-core/viralrecon workflow (https://nf-co.re/viralrecon/1.1.0 ), using the Wuhan-Hu-1 reference genome (NCBI accession NC_045512.2). Swift v2 primer sequences were trimmed prior to variant analysis from iVar version 1.3.1 (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7 ) retaining all calls with a minimum allele frequency of 0.01 and higher. Viral lineages were called using the Pangolin tool (https://github.com/cov-lineages/pangolin ) version 2.4.2, pango version 1.2.6, and the 5/19/21 version of the pangoLEARN model; based on the nomenclature system described in 59 (link).
Protocol detail
SARS-CoV-2 Genomic Sequencing from Saliva
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Corresponding Organization :
Other organizations : Los Alamos National Laboratory, University of Illinois Urbana-Champaign, University of Massachusetts Chan Medical School, Johns Hopkins University, Johns Hopkins Medicine, UMass Memorial Medical Center, UMass Memorial Health Care, National Institute of Biomedical Imaging and Bioengineering
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Variable analysis
independent variables
- Heat inactivation of saliva samples (30 minutes at 95°C)
- Viral RNA extracted from saliva samples
- Viral cDNA generated from viral RNA
- Sequencing libraries generated from viral cDNA
- Viral lineages called using the Pangolin tool
- QIAamp viral RNA mini kit (QIAGEN) used for viral RNA extraction
- SuperScript IV first strand synthesis kit (Invitrogen) used for cDNA generation
- Swift SNAP Amplicon SARS CoV2 kit with additional coverage panel and unique dual indexing (Swift Biosciences) used for sequencing library preparation
- Illumina Novaseq SP used for sequencing
- Wuhan-Hu-1 reference genome (NCBI accession NC_045512.2) used for data analysis
- IVar version 1.3.1 used for variant analysis
- Pangolin tool version 2.4.2, pango version 1.2.6, and the 5/19/21 version of the pangoLEARN model used for viral lineage calling
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