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Hi there,
I recently got asked to take a look at some miRNA data from mouse, 6 samples with 1 million reads each off a Miseq.
I am mostly interested in quantification and presence of known miRNA. When I run this through mirdeep2 I get very few hits to mature mirna, maybe 40 with 35+ having only a single read hit out of 6 samples.
Almost none of the reads align to the mm9 genome, roughly 99% fail to align in each sample, I have checked the Qc and there is plenty of Truseq adapter even some PCR primer in some samples but others seem reasonably clean. I used cutadapt to remove all adapter sequence I could but there was no improvement in overall alignment figures.
Firstly, is 1 million reads (after filtering and removing adapters its more like 600k) enough for a miRNA analysis on mouse?
Secondly, is there any reason I'm getting such low alignment figures ? Regardless of how aggressively I trim the data it does not improve alignment to the genome or alignment to miRBase. Is there something I'm missing or is it likely something went wrong with sequencing or library prep?
Thanks!
I recently got asked to take a look at some miRNA data from mouse, 6 samples with 1 million reads each off a Miseq.
I am mostly interested in quantification and presence of known miRNA. When I run this through mirdeep2 I get very few hits to mature mirna, maybe 40 with 35+ having only a single read hit out of 6 samples.
Almost none of the reads align to the mm9 genome, roughly 99% fail to align in each sample, I have checked the Qc and there is plenty of Truseq adapter even some PCR primer in some samples but others seem reasonably clean. I used cutadapt to remove all adapter sequence I could but there was no improvement in overall alignment figures.
Firstly, is 1 million reads (after filtering and removing adapters its more like 600k) enough for a miRNA analysis on mouse?
Secondly, is there any reason I'm getting such low alignment figures ? Regardless of how aggressively I trim the data it does not improve alignment to the genome or alignment to miRBase. Is there something I'm missing or is it likely something went wrong with sequencing or library prep?
Thanks!