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Read Alignment In Rna Seq

I was wondering that we we align reads to the Reference genome, multiple reads might map to the same stretch of sequence. Multiple reads in one position means that stretch of sequence is important and is expressed more in the transcriptome. But, is there any cut-off as to how many minimum reads should align to a stretch of sequence before we classify it as over expressed. How do i calculate that cutoff. I use Tophat to align the reads and then use cufflinks to study expression.

Thanks

rna seq read fpkm
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updated by Michael Dondrup Michael Dondrup • written by Shivani Grover • Shivani Grover

A cut-off is always arbitrary by definition, in short to replace arbitrary cutoff by significance, a statistical test and replicates are required, also this does only work to compare across samples not different genes in one sample. Please have a look at what has been asked first before asking very similar questions, look here for example: http://biostar.stackexchange.com/questions/tagged?tagnames=rna-seq&sort=votes&pagesize=50

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updated by Ram Ram • written by Michael Dondrup Michael Dondrup

My question was not the cutoff across samples, but the cutoff of the number of reads that map to one gene. For example if 20 reads map to one gene, and 10 reads map to other gene, 15 to another, 5 to another, then, to select highly expressed genes, where do we place the cutoff? At 15 or 10 or 5? How to decide the significance of no of aligned reads at the same position?

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written by Shivani Grover • Shivani Grover

I do unstand what you mean, but have you had a look at the links? There is currently no way to set anything than an arbitrary cutoff of coverage for this.

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written by Michael Dondrup Michael Dondrup

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