I am trying to do some quality control checks on Illumina NGS paired end reads using FastQC 0.11.5. I tried to add the primer sequences from NEBNext multiplex library prep kit to the contaminant_list file. I already notice that the file has non-unix carriage return non-printable charaters (^M or /r). Both removing and making sure that all lines have them does not change the error. I even used the orginal contaminant file I still get:
Option c is ambiguous (casava, contaminants)
Started analysis of myfile.fastq.gz
Failed to process /path/to/file/contaminant_list.txt
uk.ac.babraham.FastQC.Sequence.SequenceFormatException: ID line didn't start with '@'
Curiously the sequence file being analyzed seems to be processed okay, so it is not corrupted:
Approx 5% complete for myfile.fastq.gz
Approx 10% complete for myfile.fastq.gz
Approx 15% complete for myfile.fastq.gz
Approx 20% complete for myfile.fastq.gz
etc....
Here are the commands I am using:
fastqc myfile.fastq.gz -o /path/to/fastqc_out -a path/to/adapter_list.txt --noextract -t 6 -j /usr/lib/jvm/java-1.8.0-openjdk-1.8.0.191.b12-0.el6_10.x86_64/jre/bin/java -c /path/to/Configuration/contaminant_list.txt
Anybody know what is going on?
It never hurts to really pay more attention to the error messages folks!
FastQC does not like you to use the -c flag if you haven't used either --casava or --contaminants. Apparently -c can mean both. Replacing -c with --contaminants in the scripts makes the error go away. Hopefully this will help some aggrieved DNA jockeys out there. Happy scripting!
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