SARS-CoV-2 Genome Sequencing Protocol

Viral RNA extracted from original swab samples and virus culture using the RNeasy minikit (Qiagen) was reverse transcribed to cDNA using a LunaScript RT SuperMix kit (New England Biolabs). Primer pools targeting SARS-CoV-2 were designed using the PrimalScheme tool (66 (link)), and PCR was conducted using PhusionFlash PCR master mix (Themo Fisher). Sequencing libraries were prepared using an NEBNext Ultra II FS DNA library kit (New England Biolabs) according to the manufacturer’s instructions and were sequenced using an Illumina Miseq with a v3 sequencing kit. Raw sequence reads were trimmed, and low quality (quality score of <30) and short (<25 nt) sequences were removed using Trimmomatic (67 (link)). The trimmed sequence reads were assembled to the reference sequence (NC_045512.2) using the BWA-MEM (68 ) algorithm implemented in SAMTools version 1.8 (69 (link)). Mutation frequencies in virus populations were estimated based on minority variant calling with LoFreq (70 (link)).

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Jiang M., Kolehmainen P., Kakkola L., Maljanen S., Melén K., Smura T., Julkunen I, & Österlund P. (2021). SARS-CoV-2 Isolates Show Impaired Replication in Human Immune Cells but Differential Ability to Replicate and Induce Innate Immunity in Lung Epithelial Cells. Microbiology Spectrum, 9(1), 10.1128/spectrum.00774-21.

Publication 2021

RNeasy minikit LunaScript RT SuperMix kit PhusionFlash PCR master mix NEBNext Ultra II FS DNA library kit

Cdna Dna library Minority Populations Primer Sars cov 2 Viral rna Virus

Corresponding Organization : University of Turku

Other organizations : University of Helsinki, Turku University Hospital

Top 5 similar protocols

Variable analysis

independent variables

Primer pools targeting SARS-CoV-2 designed using the PrimalScheme tool

dependent variables

Mutation frequencies in virus populations

control variables

Extraction of viral RNA from original swab samples and virus culture using the RNeasy minikit (Qiagen)
Reverse transcription of extracted RNA to cDNA using a LunaScript RT SuperMix kit (New England Biolabs)
PCR conducted using PhusionFlash PCR master mix (Themo Fisher)
Sequencing libraries prepared using an NEBNext Ultra II FS DNA library kit (New England Biolabs) and sequenced using an Illumina Miseq with a v3 sequencing kit
Trimming of raw sequence reads, removal of low-quality (quality score of <30) and short (<25 nt) sequences using Trimmomatic
Assembly of trimmed sequence reads to the reference sequence (NC_045512.2) using the BWA-MEM algorithm implemented in SAMTools version 1.8
Minority variant calling with LoFreq to estimate mutation frequencies in virus populations

positive controls

Not specified

negative controls

Not specified

Annotations

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