SARS-CoV2 genome sequencing protocol (1200bp amplicon "midnight" primer set, using Nanopore Rapid kit) v5
Nikki E. Freed, Olin Silander
Abstract
To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit. UPDATE: The Rapid barcoding protocol is now (March 2021) available on the Oxford Nanopore website for 1-96 samples "PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021 This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick. The ARTIC method uses 400 base pair amplicons and the Oxford Nanopore Ligation barcoding kit (LSK-109). The protocol described here, dubbed the Midnight protocol, makes two important changes to the ARTIC V3 protocol: We have increased the size of the amplicon size to 1200bp. Using just 29 amplicons, the midnight primers anneal to only 4.5% of the viral genome compared to 17% or more of the genome, lowering the chance that viral mutations will cause dropouts due to primer mismatches. We use the RAPID barcode kit RBK004 (or, available as of March 2021 for 1-96 samples with the RBK110-96), which requires less time and fewer reagents than the ligation based LSK-109 protocol. The amplicons produced in this protocol can also be used for Illumina sequencing (i.e. using a transposon based shearing like Nextera or the Lotus kit https://sg.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays#resources) The original publication that describes this protocol is here along with important considerations is here: https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=true Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066. Primer sequences are here (and listed directly in the protocol) https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharing The primer scheme .bed and .tsv files necessary for the ARTIC variant calling pipeline are at Zenodo: https://zenodo.org/record/3897530#.Xv5EFpMzadY Version history: V5: Changed Reverse transcription part of the protocol to use the LunaScript™ RT SuperMix Kit in place of SSIV as it is easier, performs similarly, and is less expensive. The PCR step was changed to use the Q5®Hot Start High-Fidelity 2X Master Mix in place of the Q5 reagents individually, as this cuts down pipetting steps, performs similarly, and is similar in cost. V4: updated .bed and .tsv file link to point to Zenodo (and not google drive). V1-V3: included primers sequences in the protocol, fixed step 17.12 from elute in "molecular grade water or Elution buffer" to elute in "10 mM Tris-HCl pH 8.0 with 50 mM NaCl", as suggested on the Oxford Nanopore protocol, changed images from ARTIC protocol image to our own.