how does stranded and un-stranded quantification work?

[weasteam]

I have one stranded library and it was aligned to genome in both un-stranded (--library-type fr-unstranded) and stranded (--library-type fr-firststrand) methods (with tophat). The gene level counts were generated with easyRNASeq separately for the two different bam files.

The raw counts are different between un-stranded and stranded aligned bam files. For some genes, the reads from the un-stranded are a little bit higher than the stranded counting. I am wondering how does this happen? When easyRNASeq quantify from the stranded bam file, does it only count the reads from the correct strand and discard the reads from the other strand? I am trying to verify this by counting the reading in IGV, but failed.How does stranded and un-stranded quantification work?

[kokonech]

The protocol strand-specificity is well-explained in this post:

http://onetipperday.blogspot.de/2012/07/how-to-tell-which-library-type-to-use.html

According TopHat FAQ the library information is used when calculating jucntions:

http://tophat.cbcb.umd.edu/faq.shtml#library_type

I am not aware how the quantification with easyRNASeq works, but in general the for strand specific library the read is counted only if the alignment is concordant with transcript strand according to the protocol rule. For unstranded library the strand of the alignment in transcript does not have any restrictions. Usually gene coutning tools have an option to specify strand-specificity of the protocol. See for example, HTSeq-count.

[weasteam]

The protocol strand-specificity is well-explained in this post:

http://onetipperday.blogspot.de/2012/07/how-to-tell-which-library-type-to-use.html

According TopHat FAQ the library information is used when calculating jucntions:

http://tophat.cbcb.umd.edu/faq.shtml#library_type

I am not aware how the quantification with easyRNASeq works, but in general the for strand specific library the read is counted only if the alignment is concordant with transcript strand according to the protocol rule. For unstranded library the strand of the alignment in transcript does not have any restrictions. Usually gene coutning tools have an option to specify strand-specificity of the protocol. See for example, HTSeq-count.

Thank you kokonech, I will compare the result outputed from htseq-count and easyrnaseq.