BA.4, BA.5 primer-variant overlap update

+ some interesting new variants...


Brad Langhorst

CDC TOAST Hours - Brad Langhorst - NEB - 2022-07-01

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


Data Flow

Nightly Download

Genome Alignment

Variant Calling


Matt Campbell

Lineage Assignment

Lineage Variants

Aligned Sequences BA.5.1

Other Variants Matter for Diagnostics


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.


  • 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


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 Available online: (2020)





12 %


Many others...

Lineage Variant Overlaps

Lineage Variant Overlaps

Not All Overlaps Disrupt Coverage

But most do...

But most do...

Multiple amplicon scheme are helpful...

Lineage Variant Overlaps

Unusual Deletions

Lynne Apone, Luo Sun, Kayli Pinet

Internal sample with apparent dropout

Confusing double amplicon dropout... variants?

Consistent junctions observed in both replicate VarSkip Long libraries

Reads splice consistently (the black lines) across this 197 bp region

(∆ 27498-27697) with bbmap aligner

Changing alginer reveals other challenges


True Deletion

Addressed by also changing primer clipping method 

True Deletion

True Deletion


True Deletion

Sean's reads (showing soft clips)

Sean Seirra-Patev, Rhode Island DPH


Matt Campbell

NEB Leadership

NEBNext Team

Lynne Apone, Kayli Pinet, Luo Sun

Sean Sierra-Patev - R.I. DPH


Nicole Nichols

Chris Mason



Discussion/ Questions

Possible Topics:

  • Primer-monitor Questions
  • Are people seeing deletions in ORF7/8?
  • Express Protocol Questions

CDC TOAST Hours 2022-07-01 BA.4, BA.5 primer-variant overlap update

By Brad Langhorst

CDC TOAST Hours 2022-07-01 BA.4, BA.5 primer-variant overlap update

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