Trimming Primers from demultiplexed reads using qiime cutadapt trim-single

[bstemple (Brooke)]

Hello! I'm new to qiime2 and am trying to remove the primers from my single-end sequences using cutadapt. I have 15 samples that I've demultiplexed using the Demultiplexing and Trimming Adapters from Reads with q2-cutadapt tutorial. All my sequences should have the same 5' to 3' primer (with the barcode/adapters preceding it) and thus I am attempting to remove the primer sequence (32 bp) and all bp preceding it from the left end of my sequences, using the --p-front command. The issue I am having is when I run the cutadapt trim-single command to remove the primer sequence for each of my samples the sample summary is only identifying ~1% of reads with the primer (adapter is the word qiime uses) when it should be almost if not all of the reads in each of the 15 samples. Here is my code below:

qiime cutadapt trim-single --i-demultiplexed-sequences demultiplexed-seqs.qza --p-front AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT --p-error-rate 0 --o-trimmed-sequences Primer_trimmed-seqs.qza --verbose

Here is one of the summary outputs as an example from my first sample:

=== Summary ===

Total reads processed: 53,033
Reads with adapters: 15 (0.0%)
Reads written (passing filters): 53,033 (100.0%)

Total basepairs processed: 10,959,885 bp
Total written (filtered): 10,959,839 bp (100.0%)

I'm not getting an error message I'm just not getting the trimmed sequences that I want. I did try changing the --p-error-rate up to 3 but this didn't seem to change anything.

I apologize if my question is trivial, but would appreciate any suggestions or insights. I've looked through all the cutadapt forums and haven't found anything to pinpoint my issue. I'm using qiime2core2019-1 in a virtual box on my PC. I've provided a shareable link for my demutliplexed-seqs.qza is that helps, as it was too large to upload. I've also copied the entire output of the command (info for each of the 15 samples) in a .txt if that could possibly help.

demultiplexed-seqs.qza

qiime2 cutadapt output.txt (20.2 KB)

Thank you!!

[thermokarst]

I don’t see these primers anywhere in the reads. I looked at your provenance and noticed you used cutadapt to demux — that will remove the barcodes as part of the demux process. Depending on where these adapters were, they could’ve gotten remove, too.

[bstemple (Brooke)]

Thank you so much @thermokarst for taking the time to look at this. You’re right those primers weren’t in those reads. I needed to search for the reverse complement of the primer sequences I was given -hah:sweat_smile: - and then the cutadapt worked like a charm. For anyone’s future reference I used TagCleaner to help solve this issue and it was a nifty program. Thanks again!!