How to use only forward/reverse reads for MiSeq?

Hi All,

I am processing 10 gut microbiome sequences generated by MiSeq (pair-end). The merged read from forward and reverse reads are not in good shape, too many ambiguous bases and I want to go ahead and only use the forward reads. I was asking around to see how to use mothur command to achieve that. My labmate told me I could just use the same make.contigs commands to assemble the fastq files into one fasta file and one group file. I just have to make a copy of my forward reads and list them in place of the reverse reads in the stability.files.

However, I run into a problem: I was hoping the identical reads would overlap each other and generate only one reverse or forward reads, but it generated a end-beginning merged reads for each one of them, resulting in very long reads (~700), which is too long for V1-V3 reads. I did a simple blast for a random read and find out this (I used the read before make.contigs as subject, the merged read as query):

I am wondering, is there any parameter I can set up to make sure the reads don’t merge like this? But only over lab each other and can be considered as one read?

I would recommend using and trim.seqs instead.

mothur > - create fasta and qual file from fastq file
mothur > trim.seqs(fasta=yourForwardFasta, qfile=yourForwardQual, oligos=yourOligos, other trimming parameters) - remove barcodes and primers, create group file.

if you have a file option: - creates fasta and qual files for all the files listed in your file

create a batch file to run trim.seqs:

trim.seqs(fasta=yourForwardFasta1, qfile=yourForwardQual1, oligos=yourOligos, other trimming parameters)
trim.seqs(fasta=yourForwardFasta2, qfile=yourForwardQual2, oligos=yourOligos, other trimming parameters)
trim.seqs(fasta=yourForwardFasta3, qfile=yourForwardQual3, oligos=yourOligos, other trimming parameters)

merge.files(input=yourfastaFiles, output=final.trim.fasta)
merge.files(input=yourqualFiles, output=final.trim.qual)
merge.files(input=yourgroupFiles, output=final.trim.groups)