The k-mers in the ends of the reads could be short adapter remnants not identified by the adapter clipper. Typically they get soft-clipped by the aligner, but you should get rid of them if you use BBDuk in trimbyoverlap mode.
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Originally posted by pmiguel View PostMammals have teeny little exons spread out over 10's-100's of kilobases of the the genome. Mapping RNA (which has the introns spliced out) reads to the genome isn't a good way to determine insert size. And only getting 50% of the reads to map "concordantly" doesn't seem so bad. How is bowtie2 going to handle reads spanning a splice site?
If you want to determine your insert sizes, try aligning your reads to a long (spliced) transcript instead of genomic DNA. In my experience with the MiSeq and HiSeq, your sizes will look like all the very shortest library products were sequenced preferentially.
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Phillip
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I think the problem lies in the bowtie2 command:
-1 R1.paired.fq -2 R1.unpaired.fq
-1 R1.paired.fq -2 R2.paired.fq
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Originally posted by Brian Bushnell View PostI think the problem lies in the bowtie2 command:
Based on the filenames, it appears that you are telling it to treat read1's as pairs with each other, using the output of something like Trimmomatic that produced 4 output files. You should be doing something like this:
Although, I'm not really sure why ANY of the pairs were concordant.
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Originally posted by dpryan View PostGood catch! I share your wonderment that any were concordant. I'm also surprised it didn't throw an error that the files were of different lengths.
Anyway now I almost have my inner size and I am trying to align them by TopHat. I will update this thread by the result of my TopHat aligning.
Thanks all!
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