Diverse BA.2 and BA.5 sequences with spurious T11524C and A11537G -- primer trimming?

[AngieHinrichs]

I have noticed a pattern, almost exclusively in RKI sequences (but also in a few sequences from the Netherlands and France), of T11524C and A11537G occurring together in many different branches and sub-lineages of BA.2 and BA.5. It seems unlikely that those two mutations would co-occur dozens of times in sequences from many different lineages of BA.2 and BA.5. I collected some examples of what this looks like in a phylogenetic tree in a comment in cov-lineages/pango-designation#841. Then @JosetteSchoenma replied that this problem had been seen also in the Netherlands, but @JordyCoolen fixed it in JordyCoolen/easyseq_covid19@e241231 by adjusting a primer trimming region to start at 11520 instead of 11525.

I am hopeful that a similar fix would remove the spurious T11524C + A11537G calls in so many RKI sequences. I don't have a list of affected sequences, but could make one if that would help. A few examples:

BA.5.1:
Germany/NW-RKI-I-869542/2022 EPI_ISL_13382739
Germany/NI-RKI-I-869485/2022 EPI_ISL_13382683
Germany/NW-RKI-I-845428/2022 EPI_ISL_13239109

BA.5.2.1:
Germany/NW-RKI-I-835345/2022 EPI_ISL_13033644
Germany/NW-RKI-I-848455/2022 EPI_ISL_13241301
Germany/NW-RKI-I-799266/2022 EPI_ISL_12739153

BA.5.3.2:
Germany/SH-RKI-I-784836/2022 EPI_ISL_12674661
Germany/SH-RKI-I-823299/2022 EPI_ISL_13001607
Germany/NW-RKI-I-739643/2022 EPI_ISL_12346538

BA.2.36:
Germany/BW-RKI-I-835322/2022 EPI_ISL_13033622
Germany/SN-RKI-I-723436/2022 EPI_ISL_12192631
Germany/NW-RKI-I-690722/2022 EPI_ISL_11818175

BA.2:
Germany/BW-RKI-I-542774/2022 EPI_ISL_10032437
Germany/BW-RKI-I-542785/2022 EPI_ISL_10032456
Germany/NW-RKI-I-845418/2022 EPI_ISL_13239099

BA.2.9:
Germany/NW-RKI-I-614789/2022 EPI_ISL_11455600
Germany/NW-RKI-I-637027/2022 EPI_ISL_11480485
Germany/NW-RKI-I-644150/2022 EPI_ISL_11591637

A11537G belongs in BA.1 -- but T11524C seems to occur independently many times in the BA.1 branches of the UCSC/UShER tree as well. So this probably affects all Omicron sequences.

Thanks!

[MarieLataretu]

Hi @AngieHinrichs,

thanks for reporting this!
We are in contact with the labs.
I was able to reproduce T11524C & A11537G with raw BA.5 data and the unmodified primer file.

JordyCoolen/easyseq_covid19@e241231 suggests a primer start extension of amplicon 62 and 125.
T11524C and A11537G are not called with this modified primer file. Also, C23604A is called "homozygous" instead of "heterozygous".

So far, we saw this only with the EasySeq SC2 kit and BAMClipper (trims based on genomic coordinates).