Designing around known variants to produce a more robust SARS-CoV-2 sequencing method

Date

Brad Langhorst

SFAF 2021-  Brad Langhorst - NEB - 2021-09-28

Why does NEB Care About SARS-CoV-2 Variants?

 We want be sure that our LAMP, qPCR and sequencing SARS-CoV2 kits can be updated as new variants arise

 

Other people probably need this too... FDA guidance

Lineage Variants

Other Variants Matter for Diagnostics

Nightly Download

Genome Alignment

Variant Calling

RelationalDatabase

Matt Campbell

Variant Skip Design

Hypthesis: some genomic regions are more prone to variation than others.

Approach:

  • Identify variants seen in GISAID sequences (651,177 sequences as of 2021-03-08) seen > 2000 times
  • Mask known variant positions in reference genome with Ns
  • (bedtools maskfasta)
  • Design primers
  • Vary lengths, overlaps, GC, and variant frequency threshold (primalscheme)
  • Select best design
  • Additional primers as necessary

Varskip-long and varskip-short primers do not overlap >2500 variant sites (red) or variants from lineages of concern (yellow).

Orange ARTIC v3 primers overlap with a lineage of concern (yellow).  Many ARTIC primers overlap sites with more than 2500 variant observations (red)

Varskip Design Summary

Amplicon Summary

Selected VarSkip Short

  • Long vs. Short
    • Long : Lower "surface area", but requires more intact RNA.
    • Beta testing indicated more robust performance for short version
  • Questions
    • Will varskip avoid future variants?

Varskip Durablity

Multiplex PCR Design

Mutations

Lineage Summary

2021-05-25

2021-06-23

6%

6%

6%

40%

P.1 (gamma)

B.1.617.2 (delta)

Julia L. Mullen, Ginger Tsueng, Alaa Abdel Latif, Manar Alkuzweny, Marco Cano, Emily Haag, Jerry Zhou, Mark Zeller, Emory Hufbauer, Nate Matteson, Kristian G. Andersen, Chunlei Wu, Andrew I. Su, Karthik Gangavarapu, Laura D. Hughes, and the Center for Viral Systems Biology outbreak.info. Available online: https://outbreak.info/ (2020)

6%

2021-09-28

90%

VarSkip Results

Lynne Apone, Luo Sun, Kayli Pinet

Log scale coverage

VarSkip Clinical Results

Delta Lineage Saliva Sample By Primer Set

ARTICv3 

ARTICv4 

VarSkip 

VarSkip Clinical Results

Various Lineages

VarSkip  B.1.621

VarSkip  Delta 1

VarSkip  Delta 2

VarSkip  Delta 3

VarSkip Presence in NCBI

VarSkip Coverage in NCBI

Consensus sequences classified as B.1.617.2 (Delta) in Massachusetts (where VarSkip Short was field tested) were collected from NCBI Virus and partitioned into 2613 ARTICv3 and 1319 VarSkip Short sequences using the SRA “design” field (NEB_VarSkip_v1 = VarSkip Short, others assumed to be ARTICv3). Reads were aligned to the NC_045512.2 reference using minimap2 (minimap2 -x asm5) and visualized using IGV. Consensus sequences generated from VarSkip amplicons suffered fewer dropouts (indicated by arrows).

NCBI virus link to data

Thanks!

Matt Campbell

NEB Leadership

Eileen Dimalanta

NEBNEXT Team

NEB Early Access testers

Lynne Apone, Kayli Pinet, Luo Sun

Nicole Nichols

Chris Mason

NEB LAMP, qPCR Teams

primer-monitor.neb.com

VarSkip Sequencing

VarSkip sequencing - SFAF 2021

By Brad Langhorst

VarSkip sequencing - SFAF 2021

High mutation rates observed in RNA viruses like SARS-CoV-2 have been shown to disrupt amplification based detection and sequencing methods. To produce a more variant-resistant sequencing method, we identified all variants observed more than 1000 times in consensus sequences present in GISAID. We produced thousands of multiplex PCR designs and identified those that were least affected by known variants. During product development and scale-up, we periodically evaluated designs comparing with variants that arose during product development. NEB VarSkip designs exhibited fewer primer site variant overlaps than ARTICv4 or Midnight primer schemes.

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