hCoV-2019/nCoV-2019 Version 3 Amplicon Set
The ARTIC project released a protocol for sequencing hCoV-2019 on January 22nd(​https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w​). This protocol has
been rapidly adopted by labs worldwide and resulted in a large number of nearly complete
genomes deposited into GISAID.
In testing the protocol, several groups noticed a systematic dropping out of amplicons 18 and 76
with this protocol, resulting in no more than 98% genome coverage being achievable on the V1protocol. Whilst investigating the cause of this, Itokawa ​et al.
(​https://www.biorxiv.org/content/10.1101/2020.03.10.985150v1.full​) noted thatnCoV-2019_18_LEFT and nCoV-2019_76_RIGHT might form a dimer, and suggested analternative called 2019_76_RIGHTv2. We incorporated this suggestion in our V2 protocol
substituting nCoV-2019_18_LEFT for nCoV-2019_18_LEFT_alt2. The reason we chose to
substitute nCoV-2019_18_LEFT instead was because of the availability of a high scoringalternative from the original primer3 output, however users quickly reported that this change
caused other amplicons to drop out instead resulting in a sort of ​amplicon whack-a-mole!​ Thisillustrates the unpredictable interactions between primers within a multiplex PCR reaction.
To address this issue, we have developed a V3 protocol which tries to improve coverage inproblematic amplicons by addition of alternative primers (​alts​). To achieve this we extracted allhigh scoring primers for regions 7, 9, 14, 15, 18, 21, 44, 45, 46, 76 and 89 from the primer3
output. Of these we selected the alternative with the furthest coordinates from the original
primer (to avoid very similar primers). Rather than substituting these both are added in an
equimolar fashion to the V1 scheme which generates four possible primer combinations foreach region with alternates. This dramatically improves the performance of these weakerregions and requires only 100,000 reads per sample to achieve 50x minimum coverage acrossall regions. This change does require an analysis pipeline change to correctly trim the data at
the outermost coordinates of the primer set for each region. We are hugely grateful to John
Tyson at University of British Columbia for his efforts testing these alternate primers, working
with Richard Harrigan and his team at the British Columbia Centers for Disease Control. We arealso grateful to the groups around the world for providing feedback and data to help us refine
the amplicon scheme.
While we are satisfied with the current performance of the scheme, accumulation of genetic
diversity in hCoV-2019 over time may necessitate further changes. We are also investigating a
proposal to recover the 3’ and 5’ untranslated regions.
Generating the primer set
If you do not have any primers, the full V3 primer list is accessible via the Github repository:
https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nC
oV-2019.tsv

If you already have V1 or V2 primer sets, you can upgrade these to V3 by generating 100µM
pools of the alternates for both pools, diluting them 10µM then adding 0.45µL (pool A) and0.38µL (pool B) to the respective PCR reactions in place of nuclease-free water.
Name

PoolSequence

nCoV-2019_7_LEFT_alt0

nCoV-2019_1

CATTTGCATCAGAGGCTGCTCG

nCoV-2019_7_RIGHT_alt5

nCoV-2019_1

AGGTGACAATTTGTCCACCGAC

nCoV-2019_9_LEFT_alt4

nCoV-2019_1

TTCCCACAGAAGTGTTAACAGAGG

nCoV-2019_9_RIGHT_alt2

nCoV-2019_1

GACAGCATCTGCCACAACACAG

nCoV-2019_15_LEFT_alt1

nCoV-2019_1

AGTGCTTAAAAAGTGTAAAAGTGCCT

nCoV-2019_15_RIGHT_alt3

nCoV-2019_1

ACTGTAGCTGGCACTTTGAGAGA

nCoV-2019_21_LEFT_alt2

nCoV-2019_1

GGCTATTGATTATAAACACTACACACCCT

nCoV-2019_21_RIGHT_alt0

nCoV-2019_1

GATCTGTGTGGCCAACCTCTTC

nCoV-2019_45_LEFT_alt2

nCoV-2019_1

AGTATGTACAAATACCTACAACTTGTGCTnCoV-2019_45_RIGHT_alt7

nCoV-2019_1

TTCATGTTGGTAGTTAGAGAAAGTGTGTC

nCoV-2019_89_LEFT_alt2

nCoV-2019_1

CGCGTTCCATGTGGTCATTCAA

nCoV-2019_89_RIGHT_alt4

nCoV-2019_1

ACGAGATGAAACATCTGTTGTCACT

nCoV-2019_14_LEFT_alt4

nCoV-2019_2

TGGCAATCTTCATCCAGATTCTGC

nCoV-2019_14_RIGHT_alt2

nCoV-2019_2

TGCGTGTTTCTTCTGCATGTGC

nCoV-2019_18_LEFT_alt2

nCoV-2019_2

ACTTCTATTAAATGGGCAGATAACAACTGT

nCoV-2019_18_RIGHT_alt1

nCoV-2019_2

GCTTGTTTACCACACGTACAAGG

nCoV-2019_44_LEFT_alt3nCoV-2019_2

CCACAGTACGTCTACAAGCTGG

nCoV-2019_44_RIGHT_alt0

nCoV-2019_2

CGCAGACGGTACAGACTGTGTT

nCoV-2019_46_LEFT_alt1

nCoV-2019_2

CGCTTCCAAGAAAAGGACGAAGA

nCoV-2019_46_RIGHT_alt2

nCoV-2019_2

CACGTTCACCTAAGTTGGCGTATnCoV-2019_76_LEFT_alt3

nCoV-2019_2

GGGCAAACTGGAAAGATTGCTGA

nCoV-2019_76_RIGHT_alt0

nCoV-2019_2

ACCTGTGCCTGTTAAACCATTGA

Table 1​: Primer sequences to upgrade a V1 pool to V3.

Name

PoolSequence

nCoV-2019_7_LEFT_alt0

nCoV-2019_1CATTTGCATCAGAGGCTGCTCG

nCoV-2019_7_RIGHT_alt5

nCoV-2019_1

AGGTGACAATTTGTCCACCGAC

nCoV-2019_9_LEFT_alt4

nCoV-2019_1

TTCCCACAGAAGTGTTAACAGAGG

nCoV-2019_9_RIGHT_alt2

nCoV-2019_1

GACAGCATCTGCCACAACACAG

nCoV-2019_15_LEFT_alt1

nCoV-2019_1

AGTGCTTAAAAAGTGTAAAAGTGCCT

nCoV-2019_15_RIGHT_alt3

nCoV-2019_1

ACTGTAGCTGGCACTTTGAGAGA

nCoV-2019_21_LEFT_alt2

nCoV-2019_1

GGCTATTGATTATAAACACTACACACCCT

nCoV-2019_21_RIGHT_alt0

nCoV-2019_1

GATCTGTGTGGCCAACCTCTTC

nCoV-2019_45_LEFT_alt2

nCoV-2019_1

AGTATGTACAAATACCTACAACTTGTGCT

nCoV-2019_45_RIGHT_alt7

nCoV-2019_1

TTCATGTTGGTAGTTAGAGAAAGTGTGTC

nCoV-2019_89_LEFT_alt2

nCoV-2019_1

CGCGTTCCATGTGGTCATTCAA

nCoV-2019_89_RIGHT_alt4

nCoV-2019_1

ACGAGATGAAACATCTGTTGTCACT

nCoV-2019_14_LEFT_alt4

nCoV-2019_2

TGGCAATCTTCATCCAGATTCTGC

nCoV-2019_14_RIGHT_alt2

nCoV-2019_2

TGCGTGTTTCTTCTGCATGTGC

nCoV-2019_18_LEFT

nCoV-2019_2

TGGAAATACCCACAAGTTAATGGTTTAAC

nCoV-2019_18_RIGHT_alt1

nCoV-2019_2GCTTGTTTACCACACGTACAAGG

nCoV-2019_44_LEFT_alt3

nCoV-2019_2

CCACAGTACGTCTACAAGCTGG

nCoV-2019_44_RIGHT_alt0

nCoV-2019_2

CGCAGACGGTACAGACTGTGTT

nCoV-2019_46_LEFT_alt1

nCoV-2019_2

CGCTTCCAAGAAAAGGACGAAGA

nCoV-2019_46_RIGHT_alt2

nCoV-2019_2

CACGTTCACCTAAGTTGGCGTATnCoV-2019_76_LEFT_alt3

nCoV-2019_2

GGGCAAACTGGAAAGATTGCTGA

nCoV-2019_76_RIGHT_alt0

nCoV-2019_2

ACCTGTGCCTGTTAAACCATTGA

Table 2​: Primer sequences to upgrade a V2 pool to a V3

Expected results
On a test sample, with 100,000 reads randomly subsamples per barcode, we obtained thefollowing results on a typical clinical sample:

Figure 1:​ Comparison of V1, V2 and V3 protocols run on the same clinical sample with abarcoded library. For comparison, 100,000 randomly subsampled reads were used. As a guideto sufficient coverage, the vertical dotted line indicates 50x coverage.
Future supply
Currently oligonucleotide synthesis facilities such as IDT are working at high capacity due tohigh demand for qPCR reagents and orders may take longer than usual. We are organising abulk production of these V3 oligos via a manufacturer which will allow the supply of 100µM
pools ready to use. This has the benefits of reducing duplicate small-scale synthesis runs andmean you won’t have to pool the primers yourself. It is also becoming more difficult for us toorchestrate DHL shipments from Birmingham as collection services have now stopped. We
expect these to be available over the coming weeks. If you need to order these oligos now
please consider joining up with other people also doing ARTIC protocol sequencing to reduce
the number of orders being placed and the burden on overstretched manufacturing capacity
during the COVID-19 pandemic.
Josh Quick and Nick Loman on behalf of the ARTIC Consortium

24th March 2020