Trimmomatic - 2nd paired fastq is empty

Question

[dsyap]

Hi! I ran Trimmomatic on paired end reads which, as I understand, is supposed to yield 1_paired, 1_unpaired, 2_paired, and 2_unpaired fastq files. However, after the process was completed, my 2_paired file seemed empty, as the font of the other files were colored red while the 2nd one was colored white and when I ran a FASTQC on these files, I was only able to get reports for the other three files, but not the 2_paired file. Does this happen sometimes? Should I just proceed with mapping/alignment with just the 1_paired fastq file? Thank you!

[jv]

Are your insert/fragments short in length, as in close to or shorter than the read length?

When ILLUMINACLIP is used on paired-end data the default is to remove R2 if R2 is identical (well reverse compliment actually) to R1 in sequence. This is not well documented, but the appropriate information is available at, for instance:

IILLUMINACLIP also supports two additional optional parameters, which affect palindrome mode only.

ILLUMINACLIP:<fastaWithAdaptersEtc>:<seed mismatches>:<palindrome clip threshold>:<simple clip threshold>:<minAdapterLength>:<keepBothReads>

minAdapterLength: In addition to the alignment score, palindrome mode can verify that a minimum length of adapter has been detected. If unspecified, this defaults to 8 bases, for historical reasons. However, since palindrome mode has a very low false positive rate, this can be safely reduced, even down to 1, to allow shorter adapter fragments to be removed.

keepBothReads: After read-though has been detected by palindrome mode, and the adapter sequence removed, the reverse read contains the same sequence information as the forward read, albeit in reverse complement. For this reason, the default behaviour is to entirely drop the reverse read. By specifying „true‟ for this parameter, the reverse read will also be retained, which may be useful e.g. if the downstream tools cannot handle a combination of paired and unpaired reads.

[GenoMax]

Please post the command you are using.

[dsyap]

This was I put as part of a loop that will sort through the folders of the fastq files to be trimmed:

trimmomatic PE -threads 32 -phred33 ${input}_1.fastq.gz ${input}_2.fastq.gz ${output}_1.trimmed.paired.fastq.gz ${output}_1.trimmed.unpaired.fastq.gz ${output}_2.trimmed.paired.fastq.qz ${output}_2.trimmed.unpaired.fastq.gz ILLUMINACLIP:7_misc/TruSeqAdapter.fa:2:30:10:2:True LEADING:3 TRAILING:3 MINLEN:36