We were able to extract SARS-CoV-2 RNA from the submandibular lymph node of 1 pig (20–06), which was processed for high-throughput sequencing by CFIA National Centre for Foreign Animal Disease (NCFAD) Genomics Unit with enrichment for sequences for vertebrate viruses according to previously published method (24,25) and sequenced on a MiSeq (Illumina, https://www.illumina.com) using MiSeq Reagent Kit v3 (600-cycle; Illumina). Data analysis also were performed by the CFIA NCFAD Genomics Unit using nf-villumina version 2.0.0 (https://github.com/peterk87/nf-villumina), an in-house workflow developed by using Nextflow (26), which performed read quality filtering with fastp (27); Centrifuge version 1.0.4-beta (28) and Kraken2 version 2.0.8 (29) read taxonomic classification using an index of the National Center for Biotechnology Information (NCBI) nucleotide database (downloaded 2020 Feb 4); a Kraken2 index of NCBI RefSeq (https://www.ncbi.nlm.nih.gov/refseq) sequences of archaea, bacterial, viral, and human genomes GRCh38 (downloaded and built on 2019 Mar 22); removal of nonviral reads (i.e., not classified as belonging to superkingdom “Viruses” (NCBI taxonomic identification 10239) by using Kraken2 and Centrifuge taxonomic classification results; de novo metagenomics assembly of taxonomically filtered reads by Shovill version 1.0.9 (30), Unicycler version 1.0.9 (31), and Megahit version 1.2.9 (32); and nucleotide BLAST+ version 2.9.0 (33,34) search of all assembled contigs against the NCBI nucleotide BLAST database (downloaded 2020 April 10) using the “update_blastb.pl” script as part of the blast Bioconda package (35). We mapped nf-villumina taxonomically filtered reads against the top viral nucleotide BLAST match, SARS-CoV-2 isolate 2019-nCoV/USA-CA3/2020, MT027062.1, to generate a majority consensus sequence.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.