Bowtie2 error RNA seq aligning to genome

Question

[nidhi.vijayan13]

I first used Trimmomatic using the command:

java -jar $Trimmomatic PE ~/transcriptome/WT_LO2_1.fastq ~/transcriptome/WT_LO2_2.fastq ~/transcriptome/trimmed/WT_LO/WT_LO2_forward.fq.gz ~/transcriptome/trimmed/WT_LO/WT_LO2_reverse.fq.gz ILLUMINACLIP:TruSeq3-PE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50

I tried it with and without "keepbothreads" option. Both times I got 10x more forward reads than reverse

Then when I use bowtie to map to the genome using the script:

bowtie2 -x host_DB -1 ~/transcriptome/trimmed/WT_LO/WT_LO1_forward.fastq -2 ~/transcriptome/trimmed/WT_LO/WT_LO1_reverse.fastq -S WT_LO1_mapped_and_unmapped.sam

It starts the process and I get an output file, but it stops with an error: Error, fewer reads in file specified with -2 than in file specified with -1 terminate called after throwing an instance of 'int' (ERR): bowtie2-align died with signal 6 (ABRT)

[GenoMax]

You can try repair.sh from BBMap suite to re-sync paired end reads in the two files and remove singletons.

Since you did trim your data files together I suggest going back to the original data files and repairing those files before doing the trimming. I suspect your original files are not in sync.

Answer 1

[nidhi.vijayan13]

I fixed it by using the following script:

java -jar $Trimmomatic PE ~/transcriptome/WT_LO2_1.fastq ~/transcriptome/WT_LO2_2.fastq LO2_forward_paired.fq.gz LO2_forward_unpaired.fq.gz LO2_reverse_paired.fq.gz LO2_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE:2:30:10:2:TRUE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50

The repair.sh script did not work for my files.