SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
htseq-count paolo.kunder Bioinformatics 10 10-22-2014 05:45 AM
HTseq-count of zero halffedelf RNA Sequencing 14 04-17-2014 11:31 AM
Is my parameter choice for HTSeq-count is right? wmseq Bioinformatics 15 11-07-2013 01:24 PM
Which ID should be used for HTSeq-count? syintel87 Bioinformatics 11 02-07-2013 01:16 AM
multiBamCov or htseq-count to count read per feature ? NicoBxl Bioinformatics 1 07-03-2012 03:05 AM

Reply
 
Thread Tools
Old 08-06-2014, 03:15 PM   #1
shangzhong0619
Member
 
Location: La Jolla

Join Date: Nov 2013
Posts: 17
Default HTseq-count feature type choice for RNAseq

Hi all,
I am using HTseq-count to count reads in my bam files. I used ncbi mouse annotation file.
I wonder which feature type (third column of gff3 file) I should use?
My understanding of how htseq count reads is that if I choose 'exon', then it will count reads only mapping to exons and sum those up for a gene. If I choose 'gene', it will count all the reads mapping to introns and exons of that gene. Theoretically, for RNAseq I should choose exons, and ignore reads mapping to introns.
In my sample, I know I knock out the gene ext1. I tried both choices and used DEseq2 to do differential expression analysis. In my results which I chose the 'gene' feature, the ext1 gene was the most significant gene. However, in the results for choosing 'exon', there are over 200 genes more significant than ext1. So now I am confused, it seems that the 'gene' feature are better than 'exon'? Anyone has this situation before? thanks.
shangzhong0619 is offline   Reply With Quote
Old 08-07-2014, 01:44 AM   #2
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

You'll want to use 'exon', otherwise you increase the noise from pre-mRNAs. I'm not sure why you would be disturbed to find other genes with more significant p-values than the knocked out gene. There are a few different things that affect one's ability to determine variance and the signal level (in this case, how highly expressed a gene is) is one of them. Since you're knocking the gene out, its expression should be very low in some of your samples (I'm assuming a complete knock out rather than just hemizygous), so you'll then have less power there to begin with. If this causes some other decently expressed gene to drastically increase expression then said other gene will probably have a smaller p-value. That's not really a problem.
dpryan is offline   Reply With Quote
Old 08-07-2014, 11:24 AM   #3
shangzhong0619
Member
 
Location: La Jolla

Join Date: Nov 2013
Posts: 17
Default

Quote:
Originally Posted by dpryan View Post
You'll want to use 'exon', otherwise you increase the noise from pre-mRNAs. I'm not sure why you would be disturbed to find other genes with more significant p-values than the knocked out gene. There are a few different things that affect one's ability to determine variance and the signal level (in this case, how highly expressed a gene is) is one of them. Since you're knocking the gene out, its expression should be very low in some of your samples (I'm assuming a complete knock out rather than just hemizygous), so you'll then have less power there to begin with. If this causes some other decently expressed gene to drastically increase expression then said other gene will probably have a smaller p-value. That's not really a problem.
Hi, dpryan. Thanks for your reply. Actually my knock out is hemizygous, so in this case, it is possible to have genes more significant than the knock out gene?
shangzhong0619 is offline   Reply With Quote
Old 08-07-2014, 01:29 PM   #4
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

Sure and for the reasons that I mentioned. Remember that the p-value just relates to believability of the finding given the data. Further, changes in biology are typically non-linear, so a 50% decrease in the knocked out gene could easily lead to larger changes in others. Not to mention that a hemizygous deletion will often not halve the expression.
dpryan is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 05:53 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO