How to separate forward and backward sequences that were mixed in one fastq file

Commands in mothur
1

Hi all,

So we got the sequencing results back in test.R1.fastq and test.R2.fastq files. Instead of having all the forward sequences in R1.fastq and all the backward sequences in R2.fastq, we got two fastq files mixed with both directions of the sequences. That makes things harder in terms of make.contigs command, since it won’t let me.

Any suggestion on how to extract/separate forward and backward sequences and put them in separate files, please?

Thanks much!

2

You should be able to do make.contigs without an oligos file and then run trim.seqs with an oligos file and using the checkorient parameter to remove the primers and barcodes.

Pat

3

Dr. Pschloss, thanks much for your help. It worked for one of our dataset. However, for another dataset we got a different problem. The ffastq and rfastq contain 2005473 and 2343853 reads in each file. So mothur won’t allow me to make.contigs. I’ve tried to make.list and get.seqs, for instance, make.list(fasta=xx_forward.fastq), and got the accnos file. Then I did get.seqs(accnos=xx_forward.accnos, fasta=xx_reverse.fastq), but I got an empty files.

I’ve also tried make.file, make.groups didn’t work. I used merge.files and got a merged file containing all the information. But when I ran summary.seqs, mothur reported error with mostly 25% of the bases in sequence to be ambiguous.

We got the raw data from Mr.DNA, so we expect to have mixed forward and backward reads in a file. Normally we just make contigs and trim seqs with oligo file later. But this time we couldn’t even make contigs. Any suggestion would be appreciated :slight_smile: thanks