Question: sequence in forward seq files but not in the reverse files
gravatar for flavobacteria
5.5 years ago by
United States
flavobacteria50 wrote:

when I use the command make.contigs(file=stability1.files, processors=8)

the outcome tells that some sequences appear in the forward but not in the reverse and let me use remove:

"remove it using the remove.seqs command before proceeding"


Should be something like the following:

remove.seqs(fasta=A.forwar.fastq, fasta=A.reverse.fastq)

Anyone suggests?

sequencing snp next-gen • 2.3k views
ADD COMMENTlink modified 22 months ago by vongunte0 • written 5.5 years ago by flavobacteria50

What program are you using? Are you trying to perform an assembly?

ADD REPLYlink written 5.5 years ago by Dan D7.1k

Dan, I'm running into the same issue with my data in mothur.  I demultiplexed data that we received from Mr. DNA, and now have individual forward and reverse reads that I would like to contig together in mothur using make.contigs.  However, when I run make.contigs I get the same error as above (" in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.").  However, when I try to run remove.seqs, I am told I need an accnos file.  I am not at all certain how to generate an accnos file - any suggestions?

ADD REPLYlink written 4.7 years ago by wolfgang.rumpf10

Hi Wolfgang,

Try mothur > list.seqs(fasta=[myfasta.fa])

ADD REPLYlink written 4.7 years ago by Dan D7.1k

Hi Wolfgang, I am running through the same problem. Did you fix this?

ADD REPLYlink written 2.8 years ago by ogrecio0

Hello everyone. I am also using Mr.DNA data that was separated into forward and backward fastq files for each sample. I am trying to run make.contigs on it as well and I get the same error as flavobacteria. In the meantime, is there a solution for this problem?

When I let mothur generating a stability.files list, it created 2 different ones. One is stability.paired.files containing one single sample and another one is stability.single.files containing the rest. I assume this is somewhat connected to the problem...

ADD REPLYlink written 22 months ago by vongunte0

And this is what it sais, if I try to process all files together using make.contigs:

Processing file pair 1_S2_L001_R1_001.fastq - 1_S2_L001_R2_001.fastq (files 1 of 16)

ERROR: You have 63786 sequences in your forward fastq file, but 57141 sequences in your reverse fastq file. Please use the list.seqs and get.seqs commands to make the files match before proceeding.

ADD REPLYlink modified 22 months ago • written 22 months ago by vongunte0

Error seems to be clear. For some reason your paired data files do not have the same number of reads. You can sync the files using tool from BBMap suite. Here is a guide for how to use

ADD REPLYlink written 22 months ago by genomax91k
gravatar for Dan D
5.5 years ago by
Dan D7.1k
Dan D7.1k wrote:

It looks like you're using mothur. What the error message from make.contigs is telling you is that you have some sequence identifiers in the forward read of your paired-end data that are not found in the reverse read. The remove.seqs method will help you fix this problem. It looks like you're trying to provide a tab-delimited list of sequence files. You should iterate through that list, passing the pair of files in each line to the remove.seqs method. Then you can try again to run the cleaned files through make.contigs.

In the future, please clearly state which program you're using and a general overview of what you're trying to do if it's not obvious from the question itself. You should also choose appropriate tags. It will ensure that you get a timely, quality answer. Here's a helpful post on that subject:

How To Ask Good Questions On Technical And Scientific Forums

ADD COMMENTlink modified 5.5 years ago • written 5.5 years ago by Dan D7.1k
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