Make.contigs failed as number of forward and reverse sequences are unequal. Please let me know the commands before making contigs at this stage. I got the warning in mothur as
“Processing file pair 18J_S43_L001_R1_001.fastq - 18J_S43_L001_R2_001.fastq (files 1 of 1) <<<<<
[ERROR]: You have 99010 sequences in your forward fastq file, but 247971 sequences in your reverse fastq file. Please use the list.seqs and get.seqs commands to make the files match before proceeding.”

On which fastq do I need to run list.seqs? forward or reverse?

How to remove unpaired sequences?

Please elaborate the commands step-wise before make.contigs command.

Are you using version 1.38.1? If so and you are still having issues, here’s how to force matching reads in both files.

How to remove unpaired sequences?

mothur > list.seqs(fastq=yourForwardFastqFile) - creates accnos file containing names of sequences in forward file. Choose forward file because it has less reads.
mothur > get.seqs(fastq=yourReverseFastqFile, accnos=current) - select forward reads from reverse file. Note: New reverse file could be missing some forward reads, but this command will eliminate any reads not in the forward file.
mothur > list.seqs(fastq=yourReverseFastqFile.pick.fastq) - list sequences in new reverse file. Choose this file in case it is missing any reads from forward fastq file.
mothur > get.seqs(fastq=yourForwardFastqFile, accnos=current) - select sequences from forward file that are also in reverse file.

Forward and reverse .*pick.fastq files should now match and can be run with make.contigs.

Dear Westcott,

Thanks a lot for your helpful reply.


I’m encountering the same problem. I would like ask the following:

How can I do this with multiple fastq files?

After generating .pick.fastq files, do I have to make stability files again before proceeding with make.contigs?