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Recommended analysis pipeline
The recommended workflows for the bioinformatics analyses are provided by the ARTIC network and are documented on their web pages at https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html.
The reference guided genome assembly and variant calling are also performed according to the bioinformatics protocol provided by the ARTIC network. Their best practices guide uses the software contained within the FieldBioinformatics project on GitHub.
This workflow uses only the basecalled FASTQ files to perform a high-quality reference-guided assembly of the SARS-CoV-2 genome. Sequenced reads are re-demultiplexed with the requirement that reads must contain a barcode at both ends of the sequence (this only applies to the Classic and Eco PCR tiling of SARS-CoV-2 protocols but not the Rapid Barcoding PCR tiling of SARS-CoV-2), and must not contain internal barcodes. The reads are mapped to the reference genome, primer sequences are excluded and the consensus sequence is polished. The Medaka software is used to call single-nucleotide variants while the ARTIC software reports the high-quality consensus sequence from the workflow.
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Expected results - target coverage
The graphs below show how many hours of sequencing was required to cover the SARS-CoV-2 genome to different depths of coverage using the PCR tiling of SARS-CoV-2 with SQK-RBK110.96 protocol. Higher depths of coverage and higher numbers of multiplexed samples require a longer sequencing time. However, in most cases, 8-12 hours are sufficient to achieve full coverage of the genome.
Figure 1. Sequencing time required to cover the SARS-CoV-2 genome to different depths with increasing sample numbers per flow cell. This experiment used 1.2 kb amplicons barcoded using the Rapid Barcoding Kit 96 (SQK-RBK110.96) that were sequenced on the GridION.
Figure 2. Sequencing time required to cover the SARS-CoV-2 genome to different depths with 96 samples. This experiment used 1.2 kb amplicons barcoded using the Rapid Barcoding Kit 96 (SQK-RBK110.96) that were sequenced on the PromethION.When assessing coverage of the genome with varying numbers of viral copies as input, 1000 copies of the Twist synthetic SARS-CoV-2 RNA control were sufficient for nearly full coverage.
Figure 3. Coverage of the genome in a final consensus sequence prepared using the PCR tiling of SARS-CoV-2 using the Rapid Barcoding 96 protocol. 96 samples were sequenced on a single flow cell across an input titration gradient.