Firstly, Qiime team: thank You for this lovely tool, kind forum and helpful Youtube tutorial. I've learned much thanks to You!
Nevertheless, I come seeking Your help. Here is my problem:
I used q2-cutadapt (qiime2- 2020.8) to remove primer sequences form my reads (sequencing: 2x300 bases, primers: 341f-785r, ~466 - total amplicon length) ) in preparation for Deblurring. I decided to remove read-through primer sequences, just in case. Hence, I've run:
qiime cutadapt trim-paired \ --i-demultiplexed-sequences qiime_analysis/primary_input.qza \ --p-cores 3 \ --p-front-f CCTACGGGNGGCWGCAG \ --p-front-r GACTACHVGGGTATCTAATCC \ --p-adapter-f GGATTAGATACCCBDGTAGTC \ --p-adapter-r CTGCWGCCNCCCGTAGG \ --p-match-adapter-wildcards True \ --p-discard-untrimmed True \ --o-trimmed-sequences qiime_analysis/I_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality].qza \ --verbose > qiime_analysis/I_report_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality]
BUT! I've read on Your forum that there is an alternative way of giving the same command, namely:
qiime cutadapt trim-paired \ --i-demultiplexed-sequences qiime_analysis/primary_input.qza \ --p-cores 3 \ --p-adapter-f 'CCTACGGGNGGCWGCAG...GGATTAGATACCCBDGTAGTC' \ --p-adapter-r 'GACTACHVGGGTATCTAATCC...CTGCWGCCNCCCGTAGG' \ --p-match-adapter-wildcards True \ --p-discard-untrimmed True \ --o-trimmed-sequences qiime_analysis/II_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality].qza \ --verbose > qiime_analysis/II_report_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality]
I've tried both methods (see visualizations below):
First method: [I_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality].qzv (318.5 KB) ](http://)
Second method: II_noadapter_for_deblur_SampleData[PairedEndSequencesWithQuality].qzv (318.3 KB)
As You can see, although results are very similar they do somewhat differ in lengths and quality scores of sequences produced.
Turns out that the second method produces this warning:
WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The interpretation of what this means has changed in Cutadapt 2.0. (The 5' adapter is now no longer anchored by default.) To get results consist with the old behavior, you need to anchor the 5' adapter explicitly as in '-a ^ADAPTER1...ADAPTER2'.
Thus, I would assume that the second method is obsolete and may produce unintended results. Am I correct in my conclusion?