Warning: All reads at position 4045 in the same reverse orientation ?

[Vinoy-UKHSA]

Hello V-pipe team, Thanks for the wonderful tool. My v-pipe works fine, however I am getting this warning (Warning: All reads at position 4045 in the same reverse orientation ?) for number of position around 50, and don't know what is wrong in the dataset. Can you please explain me why I get this warning and how to rectify this ? Thanks Vinoy

[DrYak]

Hello!

If I an not wrong, this is mostly due to ShoRAH not (yet) being designed for multiplex PCR protocols, like the typical ARTIC v3/v4 that we use for wastewater.

ShoRAH assumes randomly distributed reads (e.g.: like in some fragmented protocols, that sequence 50~70bp, used for clinical swabs), so it positions its windows at regular intervals. That's not the case with the typical ~250bp pair ended sequencing that we use with SARS-CoV-2 wastewater, there all reads start at the extremities of the multiplex PCR amplicons, and end also all roughly at similar places.

This has two consequences:

• ShoRAH can place windows at position along the genome where all the reads are only R1 from one amplicon. This will trigger the message that you report, but should be problematic.
• ShoRAH can also place windows at the border between two amplicons, and no single read will occupy >85% of the windows, causing ShoRAH to complain about coverage within that window, even if there is enough coverage according to the alignment.

We have PhD and Master students working on improving ShoRAH (making it multiplex PCR amplicon aware, and correctly matching the windows to the boundaries of the amplicons - this will completely negate the problems mentionned above) which will be released in the comming weeks.