Hi to all of you,
I'm more or less brand new at bioinformatics, eager to learn, but tend to end up mostly feeling stupid.
I was given a set of miRNA paired end files (run on Illumina NextSeq 500) to identify novel miRNAs as potential biomarkers in blood. Before I started to think too much of HOW to actually do the identification and differential analysis of those sequences I read up on alignment tools. So I ended up aligning my files with Bowtie2 after trimming with cutadapt. Got an overall alignment rate of around 94% for all files.
Then I came across miRDeep2 and saw the potential with that pipeline. Since I also want to learn I tried simply to align my files with Bowtie first and thought I could convert them before feeding them into the pipeline. However, for some reason I get only 1% alignment rate for my reads. Same files I got 94% alignment rate for with Bowtie2. I tried to align just one of the paired end files and got an alignment rate of over 80%. So why can't I get the paired end files to align as nicely? Any suggestions? Now that I DO have paired end it feels like a waste to only use one of the files as "single end".
Bowtie for paired end:
Result:
# reads processed: 10229619
# reads with at least one reported alignment: 91017 (0.89%)
# reads that failed to align: 10138602 (99.11%)
Reported 91017 paired-end alignments to 1 output stream(s)
Bowtie for "single end":
Result:
# reads processed: 10229619
# reads with at least one reported alignment: 8242967 (80.58%)
# reads that failed to align: 1986652 (19.42%)
Reported 73843253 alignments to 1 output stream(s)
I have tried to adjust the parameters for -X, -I and --ff/--rf/--fr without much improvement...
I'm more or less brand new at bioinformatics, eager to learn, but tend to end up mostly feeling stupid.
I was given a set of miRNA paired end files (run on Illumina NextSeq 500) to identify novel miRNAs as potential biomarkers in blood. Before I started to think too much of HOW to actually do the identification and differential analysis of those sequences I read up on alignment tools. So I ended up aligning my files with Bowtie2 after trimming with cutadapt. Got an overall alignment rate of around 94% for all files.
Then I came across miRDeep2 and saw the potential with that pipeline. Since I also want to learn I tried simply to align my files with Bowtie first and thought I could convert them before feeding them into the pipeline. However, for some reason I get only 1% alignment rate for my reads. Same files I got 94% alignment rate for with Bowtie2. I tried to align just one of the paired end files and got an alignment rate of over 80%. So why can't I get the paired end files to align as nicely? Any suggestions? Now that I DO have paired end it feels like a waste to only use one of the files as "single end".
Bowtie for paired end:
Code:
./bowtie -n 0 -l 15 -e 80 RefCat -q -1 /pathtomystorage/trimmed/trimmed_1.fq -2 /pathtomystorage/trimmed/trimmed_2.fq -S /pathtomystorage/alignment/bowtie_alignment.sam
# reads processed: 10229619
# reads with at least one reported alignment: 91017 (0.89%)
# reads that failed to align: 10138602 (99.11%)
Reported 91017 paired-end alignments to 1 output stream(s)
Bowtie for "single end":
Code:
./bowtie -a --best -n 0 -l 15 -e 80 RefCat -q /pathtomystorage/trimmed/trimmed_1.fq -S /pathtomystorage/alignment/bowtie_alignment_1_.sam
# reads processed: 10229619
# reads with at least one reported alignment: 8242967 (80.58%)
# reads that failed to align: 1986652 (19.42%)
Reported 73843253 alignments to 1 output stream(s)
I have tried to adjust the parameters for -X, -I and --ff/--rf/--fr without much improvement...
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