NEB Positive Sample Sequencing Results

 

Date

Brad Langhorst

NGS Club -  Brad Langhorst - 2022-02-22

Technial contributions from Matt, Kayli, Luo, and Lynne

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

 

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 Skipping 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

6%

6%

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

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

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

4%

?

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).

ARTICv4

Midnight

Primer Set Performance: Omicron

VarSkip2 Performance

BA.1.1

Analysis of Results

Analysis of Results

Analysis of Results

2 new plausible transmission chains (red and blue)

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

Recent Positive Detection

Breakdown by Sex

Positivity

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

NGS Club Talk 2022-02-22

By Brad Langhorst

NGS Club Talk 2022-02-22

The precise sequence of the viruses infecting our community members can inform our response. I’ll share some results and details about the workflow we use to prepare, sequence, and reason about the viruses identified in our CLIA screening.

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