Trim.seqs to remove barcodes and primers from fast files

Commands in mothur
1

Hello,

I am preparing to submit forward and reverse fastq files to ENA. They require that 16S sequences are submitted in fastq format, without primers, linkers, barcodes, etc. Trim.seqs won’t accept fastq format and make.contigs automatically outputs a fasta file. Is there another option?

Thanks,

Claire

2

Try this:

mothur > fastq.info(fastq=forward.fastq) - create fasta and qual files for trim.seqs
mothur > trim.seqs(fasta=current, qfile=current, oligos=oligosWithforwardBarcodes&Primers, other parameters…) - remove barcodes and primers from forward files.
mothur > list.seqs(fasta=current) - list sequences that mothur was able to remove forward barcodes and primers for
mothur > make.fastq(fasta=current, qfile=current) - create forward fastq file with trimmed sequences
mothur > get.seqs(fastq=reverse.fastq) - remove any sequences that failed the forward trimming.
mothur > fastq.info(fastq=current) - create fasta and qual files for trim.seqs
mothur > trim.seqs(fasta=current, qfile=current, oligos=oligosWithReverseBarcodes&Primers, other parameters…) - remove barcodes and primers from reverse files.
mothur > mothur > list.seqs(fasta=current) - list sequences that mothur was able to remove reverse barcodes and primers for
mothur > make.fastq(fasta=current, qfile=current) - create reverse fastq file with trimmed sequences
mothur > get.seqs(fastq=foward.trimmed.fastq) - remove any sequences that failed the reverse trimming.

3

Hi,

I would also like to ask:

After you get the forward and reverse fastq files that have the primers removed, can I just proceed to make.contigs command and follow the MiSeq SOP for downstream analysis?

And is there a link on how to make oligos file?

I sent my Archaea sequences out for MiSeq sequencing, and the sequencing centre used the primers 340f - 915r. Do I need to get the primer sequences and make the oligo files myself?

Thank you!

4

After you get the forward and reverse fastq files that have the primers removed, can I just proceed to make.contigs command and follow the MiSeq SOP for downstream analysis?

Yes, you can continue with the MiSeq_SOP.

And is there a link on how to make oligos file?

http://www.mothur.org/wiki/Oligos_File

I sent my Archaea sequences out for MiSeq sequencing, and the sequencing centre used the primers 340f - 915r. Do I need to get the primer sequences and make the oligo files myself?

If your sequences have primers on them, then you will need to include those primers in the oligos file.