Fastq.info + oligo file not seperating barcodes

Hi everyone,

I’m having an issue where I run the fastq.info command with an associated oligo file, but the command is only outputting 5 files (Fasta, Qual, scrap fasta, scrap qual, scrap fastq).

The command I’m using is: fastq.info(fastq=ParentMicrosat.fastq, oligos=oligoparentmicrosat.tsv)

When the command is done running through all the sequences, its says:
“Generating parsed files… Running command: split.groups(fastq=ParentMicrosat.fastq, group=ParentMicrosat.group)
Unable to open ParentMicrosat.group. Trying mothur’s executable location ParentMicrosat.group.
Unable to open ParentMicrosat.group. Trying mothur’s tools location ParentMicrosat.group.
Unable to open ParentMicrosat.group.
Unable to open ParentMicrosat.group
You need to provide a count or group file.”

I should mention that I am running mothur on Ubuntu through a virtual box, and I had to move the virtual box to a new drive (C → D), I did this by simply cloning the virtual box, so I’m wondering if that could be a potential issue?

I’ve run this command before on a different NGS run and had no issues, the command line was the exact same, except a different fastq and oligo file, so I’m not sure why it isn’t working with the new files.

I hope you can help me!

Could you try providing the full path to the fastq file? Also, I recommend upgrading to our current version 1.47.0.

Thank you for the reply. If you mean provide the full path to the fastq file in the command, how would I go about that? If you mean, tell you where my fastq file is located, it’s in the home directory.

I have mothur on the PATH so it launches from, and used files from the home directory. I’ve never had issues with this before

I am not seeing this issue in our current version, but I suspect in the version you are running the ParentMicrosat.group file is being created in the current working directory and mothur isn’t looking for it there. By adding the full path, the group file should be created where the fastq file is.

fastq.info(fastq=/yourFullPath/ParentMicrosat.fastq, oligos=oligoparentmicrosat.tsv)

something like:

fastq.info(fastq=/Users/swestcott/Desktop/mothurInputFiles/ParentMicrosat.fastq, oligos=oligoparentmicrosat.tsv)

I will definitely give this a try this evening. In the meantime, I wanted to mention how the 5 files this command is generating (fasta, qual, scrap fasta, scrap qual, and scrap fastq) are all being created in the home directory (the same directory as the fastq and oligo files [the working directory, where in launching mother from]).

I’ll try giving the command the full path to the fastq file to see if that fixes my issue later tonight and get back to you!

I tried providing the full path to the fastq file but that also didn’t work:

fastq.info(fastq=/home/james/R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.fastq, oligos=oligoparentmicrosat.tsv)

Generating parsed files… Running command: split.groups(fastq=/home/james/R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.fastq, group=/home/james/R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group)
Unable to open /home/james/R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group. Trying mothur’s executable location R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group.
Unable to open R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group. Trying mothur’s tools location R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group.
Unable to open R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group.
Unable to open /home/james/R_2021_12_01_15_39_56_user_GSS5-0202-34-400bp_850_Nov30_2021_JamesEdelMattC.530.group
You need to provide a count or group file.

Interestingly, I tried running trim.seqs with and oligo file and allfiles=T with the single fasta and qual file fastq.info provided. This also didn’t separate by barcode. It only gave a single trim.fasta, and trim.qual file. So it must be an issue with the virtual box being cloned from the C → D drive