[MU Core]

I think we've figured out the issue with the peculiar 3' end plot. Our informatics group noticed that there was no minimum length set for trimming adapter sequence. The adapter sequence starts with “AGAT”. Thus, any sequence ending in “A” gets trimmed, any sequence ending in “AG” also gets trimmed, and so forth with “AGA”, “AGAT” and for the rest of the adapter sequence.

Assuming sequences are random:
one fourth of sequences would end in “A”
one sixteenth of sequences would end in “AG”
one sixty-fourth of sequences would end in “AGA”
one 256th of sequences would end in “AGAT”

All of these “matching” endings will be trimmed resulting in the plot characteristics previously posted.

[Brian Bushnell]

Ah, yes, that's a problem. Normally I use a minimum length of 11 to prevent that kind of issue. If you have paired reads, you can use BBDuk's tbo flag in addition to the normal sequence matching. Tbo (trim by overlap) will eliminate even 1bp of adapter sequence based on overlap rather than sequence matching, to yield very complete adapter extinction without incurring bias.