Hi,
I'm working with RNA-Seq and using bowtie and tophat to align 65bp PE reads to a reference genome. My reads were sequenced from X.laevis and I'm attempting to first map to X.tropicalis (X.laevis genome is still draft version).
After trimming and filtering my reads I am left with 31*2 = 62M reads but running samtools on my accepted_hits.bam file shows that only 12M reads have mapped in total. I'm completely confused about why the number of reads mapping is so low - I've tried fine tuning the options in tophat (-r value, -N value) and using differently trimmed reads - but have seen little improvement on 20% mapping success.
In addition almost none of my reads pair properly (samtools flagstat 'properly paired' = 0.01%).
Any help would be hugely appreciated,
Thanks
I'm working with RNA-Seq and using bowtie and tophat to align 65bp PE reads to a reference genome. My reads were sequenced from X.laevis and I'm attempting to first map to X.tropicalis (X.laevis genome is still draft version).
After trimming and filtering my reads I am left with 31*2 = 62M reads but running samtools on my accepted_hits.bam file shows that only 12M reads have mapped in total. I'm completely confused about why the number of reads mapping is so low - I've tried fine tuning the options in tophat (-r value, -N value) and using differently trimmed reads - but have seen little improvement on 20% mapping success.
In addition almost none of my reads pair properly (samtools flagstat 'properly paired' = 0.01%).
Any help would be hugely appreciated,
Thanks
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