I finally guess that Mr DNA was the company which sequenced this amplicons. Ive found this mapping file within a folder: mapping.txt (198 Bytes)
Moreover, Ive analyzed the raw sequences through FastQC, and find out this results (for the forward reads only)
So I guess Ive got a tiny barcode attatched to the forward primer and mixed orentation sequences in my sample... is that right? that might explain some problems I ran into later.
Update: Ive tried my best by following the suggestions in this discussion
Ive re-imported data now as demux-paired end sequences. Then, applied cutadapt hopefully without trouble, by running this command:
qiime cutadapt trim-paired
--i-demultiplexed-sequences demux_seqs.qza
--p-front-f CAGTTCATCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC
--p-front-r GACTACHVGGGTATCTAATCC CAGTTCATCCTACGGGNGGCWGCAG
--p-match-read-wildcards
--p-match-adapter-wildcards
--p-discard-untrimmed
--o-trimmed-sequences demux_seqs-trimmed.qza
--verbose > cutadapt-log-2.txt
Then, I dont understand wich parameters should I enter in the next step to orient the output from cutadapt:
qiime rescript orient-seqs
--i-sequences demux_seqs-trimmed.qza \ #the input will be my demux_sequences trimmed by cutadapt
--i-reference-sequences reference-sequences.qza \ #which will be my reference?
--o-oriented-seqs oriented-query-sequences.qza \ #Here I should find forward or reverse reads?
--o-unmatched-seqs unmatched-sequences.qza #What should I found in this ouput?
Thanks a lot for your help!!
By the way, I dont understand why do I have to reorient my sequences. Im working based on the assumption that forward and reverse reads do not overlap (got ~460bp fragment sequenced by Illumina MiSeq). Thus, I guess that forward reads cannot contain the reverse primer (even in the opposite orentation) because polimerase couldnt go so far. Is that right?
Update2: Ive tried my best with cutadapt by following this pipeline
https://cutadapt.readthedocs.io/en/stable/guide.html#demultiplexing-paired-end-reads-in-mixed-orientation
Then I guess my forward reads correspond to 'round1_R1.fastq' and 'round2_R2.fastq' files. My next step will be to import this files to qiime as multiplex data, so they will merge on the same file. And finally get my (really) forward reads file. Then I would follow the single end pipeline Im trying to apply since ive started my analysis haha.
Am I in the rigth direction?
Sorry about such amount or replies, (I try to work on this, not just asking). Hope this will help someone with this same issues!!