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

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

Varskip Results

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

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

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)

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