"Midnight" SARS-CoV2 genome sequencing protocol using 1200bp amplicon primer set v2 and the Nanopore Rapid library kit v1
Nikki E. Freed · 2023
This protocols is part of the ANU Biosecurity mini-research project #2 "An SARS-COV2 incursion scenario: Genomics, phylogenetics, and incursions." This mini-research project is modeled on the yearly Quality Assurance Program of The Royal College of Pathologists of Australia (RCPAQAP), we take part in together with ACT Pathology. This research project is split into two major parts, identical to how the official RCPAQAP is run every year. Part #1 is focusing on the 'wet- lab' by sequencing SARS-COV2 from real world RNA samples provided by ACT Pathology especially for our ANU biosecurity course (Thank YOU!). Here you will amplify and sequence five (5) RNA samples per research group. You will assess the SARS-COV2 genome sequences for their lineage assignments using online programs, put sequences into a global context, estimate the collection date based on genetic information, and describe mutations in the spike protein. Part #2 is focusing on the 'dry-lab' by investigating a hypothetical incursion scenario in the so-called city Fantastica. You will combine genomic surveillance of SARS-COV2 with case interview data to trace the spread into of SARS-COV2 in the community and into high risk settings. We will provide you with real publicly available SARS-COV2 genome and fantasized case interviews. You will put these two together to trace the spread and suggest potential improvements in containment strategies with a focus on high risk settings. This protocol describes the wet-lab component of Part #1. It is an adaption (fork) of a previous protocol published by Nikki Freed and Olin Silander during the early days of the pandemic here and here. They designed the protocol to enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than previous methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit. 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. You will be using the midnight primer set v2 described here and purchased here https://sg.idtdna.com/pages/products/next-generation-sequencing/workflow/xgen-ngs-amplicon-sequencing/predesigned-amplicon-panels/sars-cov-2-midnight-amp-panel. The final goal is to achieve the following: Week 7: cDNA reverse transcription of RNA into DNA. Pooled amplification of the SARS-COV2 genomes chunked into ~1200bp amplicons with non-overlapping pools A and B. Week 8: Library preparation using the Nanopore Rapid barcoding kit. Sequencing your own library on a Nanopore flongel. This protocol is applicable for week 7 and 8.