cufflinks "x" class code

wenhuang · 10-18-2010, 05:23 PM

Hi,

In the newest version of cufflinks, there is a "x" class code documented as "exonic overlap with reference on the opposite strand".

I have encountered many assembled single-exon transcripts that falled into this category. I have used unstranded RNA-Seq and have not provided any option specifying library type. I was wondering how cufflinks or cuffcompare, for this matter, determined the strand of a single exon transcript.

Thanks a lot for your help!

Wen

yfukuda · 11-09-2010, 09:50 AM

Hi,
Maybe I also have a same question.

I performed cufflinks and cuffcompare (with the latest version of 0.9.1) to analyze single-end, unstranded mRNA-seq, and I obtained a lot of 'x'-categorized isoforms, almost as many as '='.

$ grep "x" WTtranscripts.tmap |wc -l
4879
$ grep "=" WTtranscripts.tmap |wc -l
6042

I am confused about this 'x' because I think strand information of each isoform is unavailable from single-end, unstranded RNA-seq..? In this case should I ignore 'x' or should I sum up '=' and 'x' etc to count the total number?

Sorry if this question is stupid, but I appreciate someone's advice.
Thanks in advance.
yfukuda

adarob · 11-09-2010, 10:07 AM

Which version of TopHat did you use? I believe there was a bug at some point that may have caused this. If you look at your BAM file, are there XS tags for every read?

yfukuda · 11-09-2010, 10:57 AM

Hi adarob,
Thank you for your comment.

I used tophat-1.0.3 (gangcai edited version to enable --best --strata options, http://seqanswers.com/forums/showthread.php?t=4099)

I don't find XS tags for reads.
However, results from edited version of tophat look slightly different from that from latest tophat (it produces two accepted_hit.sam files in the current directly and output directly. One contains mapped result and the other is for only tags). If tophat can affect this problem, can this have something to do?

Thank you.
y

wenhuang · 11-09-2010, 07:29 PM

Hi Adam,

Glad to see this post brought your attention

.

I used Tophat 1.1.1 and Cufflinks 0.9.2.

Mine is paired end reads. I don't see XS tags for unspliced reads but I see XS tags with strand information for junction reads.

My problem is that I see many single exon transcripts assembled by Cufflinks classified as "x".

I look forward to your reply.

Thank you!

yfukuda · 11-10-2010, 12:55 AM

Hi adarob, wenhuang,

Taking closer look at my sam file, I also found XS tags for reads on splicing-junction.
Should we remove or rewrite them..?
Thank you.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
GATK "Code exception"	memento	Bioinformatics	2	02-07-2012 01:41 AM
[DESeq] invalid class "CountDataSet" object	Azazel	Bioinformatics	4	11-08-2011 01:34 AM
"allele balance ratio" and "quality by depth" in VCF files	efoss	Bioinformatics	2	10-25-2011 12:13 PM
The position file formats ".clocs" and "_pos.txt"? Ist there any difference?	elgor	Illumina/Solexa	0	06-27-2011 08:55 AM
"Systems biology and administration" & "Genome generation: no engineering allowed"	seb567	Bioinformatics	0	05-25-2010 01:19 PM

10-18-2010, 05:23 PM	#1
wenhuang Member Location: Raleigh, NC Join Date: Feb 2010 Posts: 30	cufflinks "x" class code Hi, In the newest version of cufflinks, there is a "x" class code documented as "exonic overlap with reference on the opposite strand". I have encountered many assembled single-exon transcripts that falled into this category. I have used unstranded RNA-Seq and have not provided any option specifying library type. I was wondering how cufflinks or cuffcompare, for this matter, determined the strand of a single exon transcript. Thanks a lot for your help! Wen

11-09-2010, 09:50 AM	#2
yfukuda Junior Member Location: Germany Join Date: Oct 2010 Posts: 3	Hi, Maybe I also have a same question. I performed cufflinks and cuffcompare (with the latest version of 0.9.1) to analyze single-end, unstranded mRNA-seq, and I obtained a lot of 'x'-categorized isoforms, almost as many as '='. $ grep "x" WTtranscripts.tmap \|wc -l 4879 $ grep "=" WTtranscripts.tmap \|wc -l 6042 I am confused about this 'x' because I think strand information of each isoform is unavailable from single-end, unstranded RNA-seq..? In this case should I ignore 'x' or should I sum up '=' and 'x' etc to count the total number? Sorry if this question is stupid, but I appreciate someone's advice. Thanks in advance. yfukuda

11-09-2010, 10:07 AM	#3
adarob Member Location: Berkeley, CA Join Date: Jul 2010 Posts: 71	Which version of TopHat did you use? I believe there was a bug at some point that may have caused this. If you look at your BAM file, are there XS tags for every read?

11-09-2010, 10:57 AM	#4
yfukuda Junior Member Location: Germany Join Date: Oct 2010 Posts: 3	Hi adarob, Thank you for your comment. I used tophat-1.0.3 (gangcai edited version to enable --best --strata options, http://seqanswers.com/forums/showthread.php?t=4099) I don't find XS tags for reads. However, results from edited version of tophat look slightly different from that from latest tophat (it produces two accepted_hit.sam files in the current directly and output directly. One contains mapped result and the other is for only tags). If tophat can affect this problem, can this have something to do? Thank you. y

11-09-2010, 07:29 PM	#5
wenhuang Member Location: Raleigh, NC Join Date: Feb 2010 Posts: 30	Hi Adam, Glad to see this post brought your attention . I used Tophat 1.1.1 and Cufflinks 0.9.2. Mine is paired end reads. I don't see XS tags for unspliced reads but I see XS tags with strand information for junction reads. My problem is that I see many single exon transcripts assembled by Cufflinks classified as "x". I look forward to your reply. Thank you!

11-10-2010, 12:55 AM	#6
yfukuda Junior Member Location: Germany Join Date: Oct 2010 Posts: 3	Hi adarob, wenhuang, Taking closer look at my sam file, I also found XS tags for reads on splicing-junction. Should we remove or rewrite them..? Thank you.