Brad Langhorst
Product Development Group Leader - New England Biolabs
Responding to SARS-CoV-2 Variants:
primer-monitor.neb.com and Varskip multiplex sequencing
Date
Brad Langhorst
CDC TOAST Hours - Brad Langhorst - NEB - 2021-06-24
Talk Plan
- Mention primer-monitor.neb.com
- Mention varskip multiplex PCR primers
- Express protocol introduction
- Discussion
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
They don't like sunlight
Don't get them wet
Don't feed them after midnight!
https://medium.com/@Naturalish/gremlins-three-rules-an-evolutionary-analysis-de4c4fae2785
Other people probably need this too... FDA guidance
What can we do???
Shine some light
Aligned Sequences AY.1
Lineage Variants
Other Variants Matter for Diagnostics
Data, well-organized, is light
Nightly Download
Genome Alignment
Variant Calling
RelationalDatabase
Matt Campbell
Demonstration
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
- 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
Amplicon Summary
Cost/Benefit
- Long vs. Short
- Long : Lower "surface area"
- Short : Lower consequence of failure
- Questions:
- Can we achieve good coverage with 1.4 kb amplicons in typical clinical samples?
- Ct limitations? Initial results from beta testers show no relationship between Ct and coverage
- What are the relative sensitivities of varskip-short vs varskip-long? Need more clinical results
- Need to move quickly....
- Will varskip avoid future variants?
Varskip Durablity
Multiplex PCR Design
Mutations
Increasing Lineages
2021-05-25
2021-06-23
6%
6%
6%
40%
P.1 (delta)
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)
Varskip Results
Lynne Apone, Luo Sun, Kayli Pinet
Linear
Log
Balanced Varskip Long ONT
ATCC 1986 strain, 1000 copies input, 250x data coverage
Varskip-long/Midnight 1200 Coverage
Varskip-long/Midnight 1200 Coverage
Varskip Short Coverage
Twist P.1 and B.1.351 controls look good (except at boundaries)
Express Protocol
Lynne Apone, Luo Sun, Kayli Pinet
ARTIC Workflow
ONT ARTIC Standard Workflow
ONT ARTIC Express Workflow
1ul of the targeted amplification reaction used in library prep
(total rxn 25 µl, standard input 50 ng)
ONT ARTIC Express Workflow
Illumina ARTIC Workflow
Illumina ARTIC Express Protocol
1.3 µl (FS) or 2.5 µl (UII) of the targeted amplification reaction used in library prep (total rxn 25 µl)
7.5 µl of the ligation reaction used in PCR (total rxn 35 µl (FS) or 48 µl (UII)
PCR cycles increased to 6 (standard 5)
Illumina ARTIC Express Protocol
ARTIC FS
ARTIC UltraII
Library Profile
If a peak is observed at ~ 180bp, cleanup the pool with 0.7X beads
Otherwise, short reads may dominate.
10 Copies
10,000 Copies
Thanks!
Matt Campbell
NEB Leadership
Eileen Dimalanta
NEBNEXT Team
Lynne Apone, Kayli Pinet, Luo Sun
Nicole Nichols
Chris Mason
NEB LAMP, qPCR Teams
primer-monitor.neb.com
Sequencing
Discussion/ Questions
Possible Topics:
- Primer-monitor Questions
- Can we achieve good coverage with 1.4 kb amplicons in typical clinical samples? Ct limitations?
- Initial results from collaborators with clinical samples show good performance even at higher Ct (27-32)
- Express Protocol Questions
- My taste in cinema
CDC TOAST Hours 2021-06-24 Primer Monitor, Varskip Sequencing
By Brad Langhorst
