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Old 06-30-2015, 10:08 AM   #1
imsharmanitin
Postdoc Cancer Bioinformatics
 
Location: Olso, Norway

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Default library type for single end RNA-seq data

I searched lot of threads but didn't find any concrete answer.

I have single end RNA-seq data generated by on a Hi-Seq 2000 and I am not sure if there is some strand information associated with it.


The following thread
http://seqanswers.com/forums/showthread.php?t=17788

says that when we use fr-unstranded library then the reads will be uniquely mapped to +/- strand. So does it means we should use fr-unstranded for single end reads.


However, on the other hand the following thread
http://seqanswers.com/forums/showthread.php?t=9303

says that "Illumina directional sequencing protocol only does first strand sequencing. So fr-firststrand may be the option to use for TopHat and Cufflinks"


so what is right answer?


Also how to find the stand information associated with the reads ?

Last edited by imsharmanitin; 06-30-2015 at 10:15 AM.
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Old 06-30-2015, 10:19 AM   #2
GenoMax
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Are you only asking if a particular read came from watson or crick strand or are you asking if the data you have came from a stranded library protocol?

You could simply map your data to a reference and look at it in IGV to see what strand a read came from. If you need to find if your library was made by stranded protocol then you could first ask your sequence provider what kind of protocol was used to make the libraries.
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Old 06-30-2015, 03:18 PM   #3
imsharmanitin
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i was asking about the data that came from a standard library protocol.

i asked the service provider about the protocol used to generate the reads. this is the response from them

"samples have been processed to prepare non-stranded mRNA-Seq libraries with the illumina truseq protocol and sequenced to obtain 50 bases from the forward strand (single read).
Please select fr-unstranded/fr-firststrand."

now shall i choose fr-unstranded OR fr-firststrand ?
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Old 06-30-2015, 03:34 PM   #4
GenoMax
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Since your libraries were prepared using a non-stranded protocol you should choose "fr-unstranded". (http://www.illumina.com/documents/pr...ysisTopHat.pdf)
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alignment bowtie2, illuminia, rna sequencing, single end reads, tophat 2

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