Primer Scheme Switching Considerations:

Length, and Variants, and Bears - Omicron

Date

Brad Langhorst

CDC TOAST - Brad Langhorst - NEB - 2021-12-03

NEB's Interest in 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

https://primer-monitor.neb.com/

Nightly Download

Genome Alignment

Variant Calling

RelationalDatabase

Matt Campbell

Diagnostic Tests

Charité qPCR

China CDC qPCR

CDC qPCR

N2/E1 LAMP

Proprietary Assays

...

Sequencing Methods

ARTICv3

ARTICv4

ARTICv4.1

VarSkip

Midnight

...

Other Variants Matter for Diagnostics

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. Each dropout has a bigger impact on coverage.
- Beta testing indicated more robust performance for short version, especially for marginal samples
Big Question
- Will VarSkip avoid future variants?

Lineage Summary

2021-05-25

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)

2021-09-28

Lineage Summary

2021-05-25

2021-06-23

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)

2021-09-28

100%

2021-12-02

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

VarSkip Presence in NCBI

Varskip Durablity

Multiplex PCR Design

Mutations

Varskip Durablity

Multiplex PCR Design

Mutations

Omicron

Lineage Variants

Summary of Primer Overlaps by Method

One dot per amplicon, Size=number of overlapping variants

Variant Induced Coverage Problems

"ARTIC" from South Africa by the Kwazulu-Natal Research Innovation and Sequencing Platform (KRISP) and the Centre for Epidemic Response and Innovation (CERI).