VarSkip multiplex PCR designs for SARS-CoV-2 sequencing

This repository contains reference files that describe the VarSkip primers used to amplify overlapping fragments of the SARS-CoV-2 virus.

In an attempt to reduce the impact of variants on amplification efficiency, these primers were designed using PrimalSeq after masking the SARS-CoV-2 reference sequence for SNVs observed more than 4000 times in GISAID. More details can be found in scientific presentations:

• slides from a presentation at the Sequencing to Function: Analysis and Application for the Future 2021 conference (September 2021)
• screen recording of a presentation at a CDC SPHERES meeting (June 2021).

Primer Set Name	Primer positions	Primer sequence details
VarSkip 1a	neb_vss1a.primer.bed	neb_vss1a.primer.tsv
VarSkip 2	neb_vss2a.primer.bed	neb_vss2a.primer.tsv
VarSkip 2b (2a + spike-ins)	neb_vss2b.primer.bed	neb_vss2b.primer.tsv
NEBNext SARS-CoV-2 Primers (VarSkip 2f: 2b + 20 alternate primers)	NEBNext_SARS-CoV-2_Primers.primer.bed	NEBNext_SARS-CoV-2_Primers.primer.tsv
VarSkip Long 1a	neb_vsl1a.primer.bed	neb_vsl1a.primer.tsv

VarSkip primers are commerically available at NEB.com as part of the ARTIC sequencing kits. As of 2022-02-15 VarSkip 2 primers have replaced VarSkip 1a primers in these products. As of 2022-04-12, 2 supplemental primers have been tested as spike-in additions to the VarSkip2 pools to improve coverage of amplicons 49 and 57 which are affected by common BA.2 variants. The NEBNext SARS-CoV-2 Primers set adds 20 alternate primers to VarSkip 2b. In the ARTIC schemes directory it is version V2f (--scheme-version 2f), so V2f is an alias of "NEBNext_SARS-CoV-2_Primers".

VarSkip Long primers are available as a custom product for larger volume sequencing.

The files in this repository can be used with analyis methods designed for other multiplex designs (e.g. ARTIC). To avoid miscalling or masking variants in priming regions, it's important to use primer coordinates that match the primers used during amplification (e.g. do not use ARTICv3 primer files with VarSkip libraries).

The same information is presented in multiple formats for convenience. Most workflows should use neb_vss1a.primer.bed or neb_vss2b.primer.bed.

Analysis methods known to work:

• galaxy ARTIC

  • example data and results are available via galaxy

• viralrecon (tested with illumina data)

• ARTIC field bioinformatics docs

  The ARTIC field bioinformatics pipeline is commonly used to analyize results produced on the Oxford Nanopore instruments. It requires a specific directory structure and primer naming to operate correctly. The schemes directory contains a reformatted primer annotation and reference genome that meet those constraints.

  Example commands (tune this to your instrument-specific model, primer set, and directories):

  VarSkip 1a: artic minion --threads 4 --read-file example_vss1a.fastq --scheme-directory schemes --scheme-version 1a NEB_VarSkip Example --skip-nanopolish --medaka --medaka-model r941_min_fast_g303

  VarSkip 2a: artic minion --threads 4 --read-file example_vss2a.fastq --scheme-directory schemes --scheme-version 2a NEB_VarSkip Example --skip-nanopolish --medaka --medaka-model r941_min_fast_g303

  NEBNext SARS-CoV-2 Primers (VarSkip 2f): artic minion --threads 4 --read-file example_nebnext.fastq --scheme-directory schemes --scheme-version 2f NEB_VarSkip Example --skip-nanopolish --medaka --medaka-model r941_min_fast_g303

  VarSkip 1a Long: artic minion --threads 4 --read-file example_vsl1a.fastq --scheme-directory schemes --scheme-version 1a-long NEB_VarSkip Example --skip-nanopolish --medaka --medaka-model r941_min_fast_g303

Many libraries produced using VarSkip primers are available via the NCBI SRA. e.g. SRR16375192