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Old 10-28-2014, 03:51 AM   #1
dena.dinesh
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Default creating GFF file from refernce fasta

HI,

I am working a flatworm for which the reference genome is not well characterized. I found the refernece trancriptome for my species from internet and I mapped my reads to refernce trancriptome. Now I want count the reads but I couldnt find the GFF file for this trancriptome. How can I count the reads without GFF file? is it possible genrate a GFF file for this trancriptome.

Kindly guide me
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Old 10-30-2014, 04:29 PM   #2
sdriscoll
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I'm assuming you're dealing with some type of standard RNA-seq (illumina) type data. Typically when people use a GFF file to count reads the alignments were made against a genome and not a transcriptome. Because of the ambiguous nature of transcriptomes I recommend looking into RSEM, Sailfish or eXpress for your read count/expression quantification. Each of those software use an EM based algorithm to disambiguate the assignment of reads to each transcript. This seems to work fairly well even when dealing with fairly complex gene loci (i.e. many alternative isoforms). Be warned that what this will produce is isoform level expression so if your reference transcriptome contains multiple isoforms per gene there will be some additional uncertainty in this output. eXpress is nice in that it will generate a confidence interval on the FPKM values it produces so you can get an idea of those transcripts that it cannot confidently call. To the best of my knowledge the art of differential expression at the isoform level has not been fully developed though the EBSeq package claims to be able to do it.
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