Counting reads in miRNA experiment
Dear all,
in our lab we are running an Illumina small RNA protocol on human sample to collect miRNA sequence and I am running the bioinformatics part.
During library preparation we are using kit that are unstreanded. After sequencing (Illumina single read) for each sample I got the majority of reads with 5-3' orientation (about 90% of the reads) and "few" reads with orientation 3'-5'.
I have cleaned my raw data removing low quality reads, illumina adapter, reads longer than 27 nt and reads shorter than 17 nt after adapter removing. I aligned to human genome and now I need to assign and count my reads to microRNA. For doing that I am using FeatureCounts (suite subread) and I am using the gff file downloaded from mirBase. I have a doubt in summarizing the reads: do I need to count the reads independently from their orientation? Do I need to sum the reads with orientation 5'-3' and 3'-5'? Or do I only need to count the reads with 5'-3' orientation?
Many thanks,
Sara
Dear all,
in our lab we are running an Illumina small RNA protocol on human sample to collect miRNA sequence and I am running the bioinformatics part.
During library preparation we are using kit that are unstreanded. After sequencing (Illumina single read) for each sample I got the majority of reads with 5-3' orientation (about 90% of the reads) and "few" reads with orientation 3'-5'.
I have cleaned my raw data removing low quality reads, illumina adapter, reads longer than 27 nt and reads shorter than 17 nt after adapter removing. I aligned to human genome and now I need to assign and count my reads to microRNA. For doing that I am using FeatureCounts (suite subread) and I am using the gff file downloaded from mirBase. I have a doubt in summarizing the reads: do I need to count the reads independently from their orientation? Do I need to sum the reads with orientation 5'-3' and 3'-5'? Or do I only need to count the reads with 5'-3' orientation?
Many thanks,
Sara
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