I have searched if any topic related to my problem existed but didn’t find any. Sorry if I missed it.
I have illumina pair-end fastq files (using V1-V3), so it is 2 separated files. Using make.contigs(ffastq=file1.fasq, rfastq=file2.fastq), i make the overlapping and then with trim.seqs i remove the primers. i used screen.seqs to filter the contigs (maxambig=0, minlength=450, maxlength=550)… And unique.seqs function, to have only unique sequences.
Before to align my contigs, i use the database silva.nr_v123.align. For this, i decide to use pcr.seqs in order to work only with the region V1-V3. Here is the command
My oligo file looks like this :
primer AGAGTTTGATCCTGGCTCAG ATTACCGCGGCTGCTGG
My primers are needed to amplify V1-V3.
forward reference : 27f
reverse reference : 534r
However i only have 2 seqs left after this, I don’t get it at all. Does it mean my primers are not present in the databse ? How is it possible ?
Thanks to help me out with this.