Alignment/transcriptome assembly/differential expression analysis with 40bp reads?

heytreeful · 10-17-2011, 11:11 AM

Hi everyone,

I am preparing to analyze some 40bp RNA-seq data generated by Illumina HiSeq from mouse samples. It seems that most RNA-seq tools nowadays are optimized for long sequence reads (>75bp) and paired-end reads.
Can I still use TopHat and Cufflinks for these 40bp reads? Is it possible to perform transcriptome assembly with limited splice reads? Since the mouse genome is well annotated, can I get by without transcriptome assembly since the goal of our study is not to discover novel genes but just to detect differential expression of known genes? I am new to this; any suggestions/comments will be much appreciated! Thank you.

sqcrft · 03-11-2013, 06:33 AM

Hi, fellow, is there any progress? I am stuck in the same dilemma.
The read length is too short, and we need to identify some novel transcripts.

heytreeful · 03-11-2013, 09:31 AM

hey, yup you can still use the conventional analyses suite for 40bp reads. There are still some splice reads present to allow for junction identification and transcript construction. Surely the resolution wasn't as great as longer read runs, but it was sufficient for our research.

sqcrft · 03-11-2013, 09:44 AM

thanks! That is really comforting.
I had worried that the data and money might be wasted.

heytreeful · 03-11-2013, 09:54 AM

it allowed us to detect presence of novel transcripts, but it didn't allow us to characterize them very well tho. Don't worry your data are not wasted

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10-17-2011, 11:11 AM	#1
heytreeful Junior Member Location: Boston Join Date: Oct 2011 Posts: 6	Alignment/transcriptome assembly/differential expression analysis with 40bp reads? Hi everyone, I am preparing to analyze some 40bp RNA-seq data generated by Illumina HiSeq from mouse samples. It seems that most RNA-seq tools nowadays are optimized for long sequence reads (>75bp) and paired-end reads. Can I still use TopHat and Cufflinks for these 40bp reads? Is it possible to perform transcriptome assembly with limited splice reads? Since the mouse genome is well annotated, can I get by without transcriptome assembly since the goal of our study is not to discover novel genes but just to detect differential expression of known genes? I am new to this; any suggestions/comments will be much appreciated! Thank you.

03-11-2013, 06:33 AM	#2
sqcrft Member Location: boston Join Date: May 2012 Posts: 29	Hi, fellow, is there any progress? I am stuck in the same dilemma. The read length is too short, and we need to identify some novel transcripts.

03-11-2013, 09:31 AM	#3
heytreeful Junior Member Location: Boston Join Date: Oct 2011 Posts: 6	hey, yup you can still use the conventional analyses suite for 40bp reads. There are still some splice reads present to allow for junction identification and transcript construction. Surely the resolution wasn't as great as longer read runs, but it was sufficient for our research.

03-11-2013, 09:44 AM	#4
sqcrft Member Location: boston Join Date: May 2012 Posts: 29	thanks! That is really comforting. I had worried that the data and money might be wasted.

03-11-2013, 09:54 AM	#5
heytreeful Junior Member Location: Boston Join Date: Oct 2011 Posts: 6	it allowed us to detect presence of novel transcripts, but it didn't allow us to characterize them very well tho. Don't worry your data are not wasted