How to distinguish Stand/Unstrand?

[Lei-Tian]

I am using STAR to align my RNA-Seq data. How to know my RNA-seq is Stand/Unstrand? Thanks!

[DarioS]

The fastest and easiest way is to view the alignments in the BAM files using IGV. Ensure that you have first read colouring activated by right-clicking on each BAM file track then sensure that Colour Alignments By -> First-of-Pair Strand is selected. Below is a screenshot of two samples that are stranded and two that are not belonging to a publicly available cancer RNA sequencing dataset. Note how each gene has approximately half blue and half red reads for the unstranded samples. The authors misleadingly did not explain their usage of different RNA-seq kits in the journal article's Methods section which causes reanalysis problems if not identified and specially handled.

[alexdobin]

Hi Lei-Tian,

great suggestion from Dario - you can use STAR to generate the wiggle files.
Another (less visual) possibility is to run STAR with the --quantMode GeneCounts option, and count total read counts on genes in the 3 and 4 columns (these column represent different library strandedness). For stranded data one of the columns should be much larger than the other. You can use this to estimate strandedness of the data quantitatively.

Cheers
Alex

[Lei-Tian]

Hi Alex, Thanks a lot! It helped me out! Best, Lei

…

________________________________ From: alexdobin <notifications@github.com> Sent: Tuesday, April 25, 2017 12:10:07 PM To: alexdobin/STAR Cc: Lei Tian; Author Subject: Re: [alexdobin/STAR] How to distinguish Stand/Unstrand? (#258) Hi Lei-Tian, great suggestion from Dario - you can use STAR to generate the wiggle files. Another (less visual) possibility is to run STAR with the --quantMode GeneCounts option, and count total read counts on genes in the 3 and 4 columns (these column represent different library strandedness). For stranded data one of the columns should be much larger than the other. You can use this to estimate strandedness of the data quantitatively. Cheers Alex — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<#258 (comment)>, or mute the thread<https://github.com/notifications/unsubscribe-auth/AXK5tdSJMxFSxZDgs31hKFw1l4O0a7NMks5rzkUPgaJpZM4NE5h3>.

[DarioS]

If your issue has been resolved, then please close it.

[Lei-Tian]

Yes thanks! What should I close? Lei Sent from my iPhone On 5 May 2017, at 3:00 AM, DarioS <notifications@github.com<mailto:notifications@github.com>> wrote: If your issue has been resolved, then please close it. — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<#258 (comment)>, or mute the thread<https://github.com/notifications/unsubscribe-auth/AXK5teVkOjtfTWlHFBgRxH6rJP1Aur4Gks5r2vMmgaJpZM4NE5h3>.

[Lei-Tian]

Already done, thanks! Lei On 5 May 2017, at 3:00 AM, DarioS <notifications@github.com<mailto:notifications@github.com>> wrote: If your issue has been resolved, then please close it. — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub<#258 (comment)>, or mute the thread<https://github.com/notifications/unsubscribe-auth/AXK5teVkOjtfTWlHFBgRxH6rJP1Aur4Gks5r2vMmgaJpZM4NE5h3>.

[xinl22]

@alexdobin
Hi Alex, if I have stranded reads and run STAR with the --quantMode GeneCount, for a specific gene, which value should I use to represent gene expression level in following analysis? The 3rd or 4th column?