pcr.seqs / trim.seqs

Commands in mothur
1

Greetings,

I am trying to remove my primer pairs from my sequenced data. I have my oligos file in the correct format (I think):
forward GGACAGAAAGACCCTATGAA
#reverse CTCTAGGGATAACAGGCTGA

However, when I input the command, it only removes the first 8 bp; not the full 20 bp. Is there anyway to fix this?

Thanks

1 Like
2

Also, sorry for the double question, how do I go abouts to remove the reverse primer sequence?

Thanks!

3

Mothur has several ways to remove paired primers from your sequences. Here’s a link to mothur’s oligos file format, http://www.mothur.org/wiki/Oligos_File.

Trim.seqs - http://www.mothur.org/wiki/Trim.seqs
The trim.seqs command can be used to remove paired or unpaired primers from assembled reads in fasta form. Your oligos file would look like:

primer GGACAGAAAGACCCTATGAA CTCTAGGGATAACAGGCTGA

mothur > trim.seqs(fasta=yourFasta, oligos=yourOligos, otherparameters…)

Make.contigs - http://www.mothur.org/wiki/Make.contigs
The make.contigs command can be used to remove paired primers and assemble paired reads in fasta or fastq form.

Your oligos file would look like:

primer GGACAGAAAGACCCTATGAA CTCTAGGGATAACAGGCTGA

mothur > make.contigs(ffastq=yourForwardFastq, rfastq=yourReverseFastq, oligos=yourOligos, otherparameters…)

Pcr.seqs - http://www.mothur.org/wiki/Pcr.seqs
The pcr.seqs command is designed to be used to trim sequences to a given region using primers. Mothur will scan the sequence looking for the primers and remove or blank out the sequence up to the point the primers are found. Your oligos file can look like:

primer GGACAGAAAGACCCTATGAA CTCTAGGGATAACAGGCTGA

or

forward GGACAGAAAGACCCTATGAA
reverse CTCTAGGGATAACAGGCTGA

mothur > pcr.seqs(fasta=yourFasta, oligos=yourOligos, otherparameters…)

Kindly,
Sarah

4

Hi Sarah,

Thank you for writing those suggestions. I have already tried those. I have no problem removing the forward primer, but mothur does not seem to like to remove the reverse primers.

If you ask them to remove both primers at once, it scraps all your sequences. So I remove the forward primer first, however I cannot seem to remove the reverse.

5

So I used
forward GGACAGAAAGACCCTATGAA
#reverse CTCTAGGGATAACAGGCTGA

I had to use the # because I read that if you don’t use it, all your sequences gets sent to the scrap file.

Then when I reran the oligo file but put the # on the forward primer, it did not remove the reverse primer.

In addition, if I used the

primer GGACAGAAAGACCCTATGAA CTCTAGGGATAACAGGCTGA

All my sequences get sent to the scrap file.

6

What kind of sequence data are you working with (e.g. 454, MiSeq) and how were they generated?

If these are 454, can you post an example of a sequence and if it’s MiSeq can you post an example of a forward sequence and a reverse sequence?

Pat