The ARTIC workflows with Illumina / short read sequencing

Dear all,

For Virus sequencing do you know of any activities to use your V3 priming set with Illumina? We started working on it and would be interest in exchanging ideas.

Cheers and many thanks in advance!!!

Andreas

Try NEB Kit E7645 ligation protocol or illumina Nextera XT (minimal 300bp amplicon, but hard to trim primers in bioinformatics)

I’m aware of a couple of protocols that can use the ARTIC amplicons to make Illumina libraries:

A full-length ligation-based one from Nate Grubaugh’s lab (via http://grubaughlab.com/open-science/amplicon-sequencing/):
https://docs.google.com/document/d/1PilT4w5jHO-ROsE8TL5WBGa0wSCdTHAsNl1LIOYiTgk/edit

Original version in our Nature Protocols paper:
https://www.nature.com/articles/nprot.2017.066

An enzymatic based one (QIAseq FX) from Itokawa’s lab:

I have also heard of people using Nextera XT with success but not sure if there’s a published protocol.

I will update this page if I hear of others, or please post below!

QIASeq FX works good for us so far.

We also use in-house script to trim primer parts.

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This protocol by JS Eden uses some of the Artic primers to generate 2.5 amplicons that are sequenced on an Illumina platform using the Nextera XT library prep. NEAR-WHOLE GENOME SEQUENCING OF SARS-CoV-2 USING POOLED AMPLICONS ON ILLUMINA PLATFORMS

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Does the step 1 start as reverse PCR, because I recognized you started with viral RNA?

Hi Nick
I tried V3 version primer set (including alt primers) and we observed a very low coverage at positions 64, 66, 70 and 74 in 50% of my samples with read counts of >180,000. We are using the same method of cDNA prep and pool amplification as Arctic protocol however we used Nextera kit for library preparation. I was wondering if you can advise if this problem is because of library prep methodology or new primer dimer formation.

We made a 24-plex plexWell library using the ARTIC workflow with the Twist synthetic RNA SARS-CoV-2 controls as input into the RT:


The gaps in the coverage correspond to the breakpoints in between the Twist synthetic constructs (non-overlapping).

Here are more data from our plexWell pilot study with the ARTIC protocol and sequencing as a 24-plex on a MiSeq Micro v2. Scaling the protocol up to 100’s of samples per day underway:



plexWell-384 library prep kits are available for evaluation from seqWell.

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Nice to see your results. How was the distribution of insert lengths?

Here is a slice of insert size data from a 96-well plate:


There was very consistent insert sizes across all samples regardless of input into the RT reactions. Uniform insert size distribution is a feature of plexWell technology. Thank you for the question! -Jack-

We used the improved ARTIC v3 primers from your publication (Itokawa_2020_A proposal of an alternative primer for the ARTIC Network’s multiplex PCR to improve coverage of SARS-CoV-2 genome sequencing). Our uniformity of coverage and alignment rate looked quite good except for the gaps due to the breakpoints in the synthetic Twist RNA controls (we are only a BSL-1 lab). On another forum, a researcher has asked whether the results from the ARTIC protocol would have been as consistent with actual clinical samples, which seems like a legitimate concern. Do you have any comments that I could share with this researcher to help address his concerns? Thanks, -Jack-

Is ARTIC v3 you mean is the primer set with 22 spike-in primers? That is not a primer set designed by us. That was designed by ARTIC Network. We have not tested the V3 primer set, yet, so I can not comment about it precisely.

With the primer set that we designed (including 6 primer exchanges: https://github.com/ItokawaK/Alt_nCov2019_primers), the results actually vary on clinical samples. Ct value seems the main factor for this variation. Samples with high Ct value tend to require high average coverage to recover weak amplicons. Nevertheless, most samples with Ct-value under 33 could be sequenced whole genome with 50 Mb / sample sequencing effort.