Brad Langhorst
Product Development Group Leader - New England Biolabs
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.
