How to count aligned RNA-seq reads after sequenced and aligned by Illumina?

IceWater · 04-03-2012, 04:02 PM

Hello All,

Illumina (using CASAVA 1.8.2) sequenced and aligned my RNA-seq samples, but the count was not done. I searched the Internet, but didn't get the way to do the count. If any of you know how to do it, could you kindly tell me. Any hint will be much appreciated.

NextGenSeq · 04-04-2012, 02:06 PM

Nobody uses the Illumina software for alignment. Get the FASTQ files and re-align with BWA, Bowtie or something else.

IceWater · 04-04-2012, 05:02 PM

Hi NextGenSeq,

Thank you very much for your reply. Is there any reason that nobody uses the Illumina software for alignment? I am a newbi in this field, and thought it would be fine to use the alignment from Illumina.

faozhi · 04-05-2012, 05:36 AM

You have to map the reads to the reference genome. To count, you might want to try cufflinks.

westerman · 04-05-2012, 06:47 AM

Different people do like different tools. Personally I use Tophat then Cufflinks/cuffdiff for RNA-seq samples against a known reference. There does seem to be the feeling that the 3rd party tools, perhaps because they are more open, are better.

IceWater · 04-05-2012, 10:18 AM

Hi faozhi & westerman, thank you very much. I will find out how to use the tools you suggested.

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04-03-2012, 04:02 PM	#1
IceWater Junior Member Location: USA CA Join Date: Apr 2012 Posts: 7	How to count aligned RNA-seq reads after sequenced and aligned by Illumina? Hello All, Illumina (using CASAVA 1.8.2) sequenced and aligned my RNA-seq samples, but the count was not done. I searched the Internet, but didn't get the way to do the count. If any of you know how to do it, could you kindly tell me. Any hint will be much appreciated.

04-04-2012, 02:06 PM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	Nobody uses the Illumina software for alignment. Get the FASTQ files and re-align with BWA, Bowtie or something else.

04-04-2012, 05:02 PM	#3
IceWater Junior Member Location: USA CA Join Date: Apr 2012 Posts: 7	Hi NextGenSeq, Thank you very much for your reply. Is there any reason that nobody uses the Illumina software for alignment? I am a newbi in this field, and thought it would be fine to use the alignment from Illumina.

04-05-2012, 05:36 AM	#4
faozhi Junior Member Location: Australia Join Date: Dec 2011 Posts: 5	You have to map the reads to the reference genome. To count, you might want to try cufflinks.

04-05-2012, 06:47 AM	#5
westerman Rick Westerman Location: Purdue University, Indiana, USA Join Date: Jun 2008 Posts: 1,104	Different people do like different tools. Personally I use Tophat then Cufflinks/cuffdiff for RNA-seq samples against a known reference. There does seem to be the feeling that the 3rd party tools, perhaps because they are more open, are better.

04-05-2012, 10:18 AM	#6
IceWater Junior Member Location: USA CA Join Date: Apr 2012 Posts: 7	Hi faozhi & westerman, thank you very much. I will find out how to use the tools you suggested.