Talk Plan

Review primer-monitor
Describe lineage variant approach
VarSkip review and status update
BA.4, BA.5, BA.5.1 update
Large deletions in ORF7a
Discussion

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

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

Other people probably need this too... FDA guidance

response: primer-monitor.neb.com

Data Flow

Nightly Download

Genome Alignment

Variant Calling

RelationalDatabase

Matt Campbell

Lineage Assignment

Lineage Variants

Aligned Sequences BA.5.1

Other Variants Matter for Diagnostics

Primer-monitor

Planned Features

More geographic detail ✅
Fully automated intake/update ✅
Automated visualization updates ✅
Automated notification ✅
Primer set scores ✅
- View primer set as a whole
- Map based visualization
Variants by time
Extensions for large primer sets
Automated lineage calling
- recording lineage history for each sequence
Suggestions welcome!

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

VarSkip Variant Response Approach

Add spike-in primers (available at no cost upon request) as new variants arise
- e.g. VarSkip v2b in January (additional primers can be spiked in by customers)
When spike-ins are insufficient, redesign, QC large scale oligo synthesis, reformulation, revalidation, and release a new revision (with notification by production change)
- e.g. VarSkip 1 -> VarSkip 2 (last fall)

Varskip Durablity

Multiplex PCR Design

Mutations

Increasing Lineages

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)