Issue with Primer Removal- Residual Primers After Cutadapt

[alannajmcc16]

Context: I'm very new to DNA sequencing and bioinformatics, and I'm currently following the DADA2 tutorial for primer identification and removal. My sequence data was generated using Illumina Miseq 2x300 paired-end sequencing, targeting the V3-V4 region, with the primer set FWD: "CCTACGGGNGGCWGCAG" and REV: "GACTACHVGGGTATCTAATCC"

Issue: During the primer identification step, i observed the following results: Forward Complement Reverse RevComp FWD.ForwardReads 135310 0 0 0 FWD.ReverseReads 9 0 0 15 REV.ForwardReads 12 0 0 22 REV.ReverseReads 132023 0 0 0

The high counts for FWD.ForwardReads in the forward orientation and REV.ReverseReads in the reverse complement orientation are as expected. However, I'm seeing small numbers of reads in FWD.ReverseReads and REV.ForwardReads (i.e., 9 and 12). is it common to have such small counts here, or does this indicate a potential issue?

After running Cutadapt to remove the primers, most of the primers were successfully removed, but a small number remained: Forward Complement Reverse RevComp FWD.ForwardReads 0 0 0 0 FWD.ReverseReads 9 0 0 0 REV.ForwardReads 12 0 0 0 REV.ReverseReads 0 0 0 0

Should i be concerned about these residual primers? is there a way to modify my approach or the code to ensure complete removal of these primers before a continue further?

Any guidance or suggestion would be greatly appreciated! :)

[benjjneb]

Should i be concerned about these residual primers?

No. That is a very small number of primers remaining. You've removed 99.99% of the primers successfully.

[alannajmcc16]

Thank you for the quick response, really appreciate it!!