when is the best step to remove my primers sequence?
Would there be any problem to take them off after classify.seqs step?
Thank so much
are you sure you have sequencing primers? if you use kozich or caporasso primers, you don’t have have sequencing primers.
I used capotado reference
However I’ve double check on fasta file and both primers are still there…
**caporaso as reference
I was evaluating my files a few minutes ago and I’ve observed something strange regarding primers removing.
Both primers (forward and reverse) were present in the file sample.trim.contigs.good.unique.fasta", however in the next one “gavião.trim.contigs.good.unique.good.filter.fasta” they were no longer there; It is important to say that I didn’t include at any command line neither primers’ sequence nor oligos file, for example.
Could anyone explain me whether mothur 1.43 is able to recognise primers sequence around this step?
If thery’re there then you can remove them in make.contigs or with trim.seqs using the oligos option.
I’ve done that, I took my fasta and count file plus oligos and applied a trim.seqs command. however all sequences were removed…
maybe should I restart the analysis and take primers off (by trim.seqs) after contigs already ready?
Just my two cents:
If you are aligning them against a good database, I remove them after the align.seqs, checking where in the alignment my fragment starts / ends.
If they were all removed then you either didn’t have primers there in the first place or your primer sequence is wrong
The problem with this approach is that if the barcodes are also still attached then you won’t be able to assign the sequences to groups. It will also massively inflate the number of sequences because there are likely to be duplicate sequences across samples.
I’ve double check on my raw sequences and there are just forward and reverse primers … they are the first sequence on my files, then indexes and barcodes were removed after sequencing surely
I do it after I demultiplexed (so barcodes are not there), sorted all them into different samples, Q trimmed, etc, right before remove chimeras. All are assigned to samples. After that, unique - then chimera removal? I did this once when the sequences came to me from a different project and I think they used the nextera instead of the cheap 2-step PCR, and I discovered after alignment that the primers were there…
If you can post an example pair of sequences with your primers we can take a look