I am trying to use mothur to run analysis of a set of community samples. I have paired end reads for multiple samples in *.fastq format. I am getting an error message “Error opening file: No such file or directory” while running make.contigs, but make.contigs still runs and this this message doesn’t seem to be related to any of the input files I’m using.
I’m using make.contigs to combine the forward and reverse reads, with the oligos option to trim off primer sequences. The command I’m running is:
mothur > make.contigs(file=fonseca.files, oligos=fon.contigs.oligos, checkorient=T, processors=20)
fonseca.files is in 3-column format, with sample ID in the first column, location of the forward fastq file in the second column and location of the reverse fastq file in the third column, as so:
FA3_N3_0.5 ~/NIGHT_TOW_Fonseca_Machida12S/1A_S1/R1.Fonseca.fastq ~/NIGHT_TOW_Fonseca_Machida12S/1A_S1/R2.Fonseca.fastq
FA3_N3_0.2 ~/NIGHT_TOW_Fonseca_Machida12S/1B_S2/R1.Fonseca.fastq ~/NIGHT_TOW_Fonseca_Machida12S/1B_S2/R2.Fonseca.fastq …
My oligos file is formatted as follows (sequences were previously demultiplexed using both reverse and forward primers, so some have the forward primer left and some of the reverse primer left):
primer GCTTGTCTCAAAGATTAAGCC NONE V3
primer NONE GCCTGCTGCCTTCCTTGGA V3
I have tried running make.contigs without the oligos option, without checkorient, with only 1 processor, and with direct input of the forward/reverse files (rather than the *.files option). None of these make any difference. When I change the input reference to an incorrect file (or one that doesn’t exist), the error message changes to “Unable to open incorrect file name [ERROR]: did not complete make.contigs”.
I noted in the MiSeq SOP that make.contigs first processes the fastq files to generate individual fasta and qual files, then makes contigs from these intermediate files. This leads me to believe that the error is due to some internal intermediate file. Can anybody corroborate this theory? Or is there some simple input I’m missing?
I am running this on my university’s computing cluster using a slurm scheduler. Is it possible that intermediate files are being placed in a different directory and mothur cannot find them?
Below is the output from running the command:
mothur > make.contigs(ffastq=~/NIGHT_TOW_Fonseca_Machida12S/1A_S1/R1.Fonseca.fastq, rfastq=~/NIGHT_TOW_Fonseca_Machida12S/1A_S1/R2.Fonseca.fastq)
Using 1 processors.
Error opening file: No such file or directory
It took 71 secs to process 65811 sequences.
Output File Names: