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Old 07-24-2013, 01:26 PM   #1
afkoeppel
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Location: VA

Join Date: Jul 2011
Posts: 17
Default Annotation of Mouse Rbpj gene

Hello everyone,

I'm analyzing an RNA-Seq experiment and I'm looking for some advice about how to interpret an anomaly that I'm seeing.

I opened up the alignments for each of my samples in IGV, because I wanted to look at the alignment for the Rbpj gene, a gene that I'm interested in, but for which I didn't get quite high enough coverage to analyze.

I noticed I had a number of reads hit the same location between exons 1 and 2 in all of my samples, but that that region was not annotated as an exon (see attached screen shot).

I checked out the annotations for the gene on ...

UCSC: http://genome.ucsc.edu/cgi-bin/hgTra...ches=NM_009035,

... and Ensembl: http://useast.ensembl.org/Mus_muscul...55779-53657445

In both cases, it appears that there is an exon (or something) between the first and second exons of Rbjp (right where my reads are hitting) in some entries, but not in others.

I'm looking for some help in figuring out what to make of this. Is this a real exon that didn't make it into the mm9 annotation?

Thanks,

Alex

Some additional Info:
  • Illumina paired-end reads
  • Aligned reads to mm9 with STAR 2.3.0
  • I used HTSeq-count to count the reads mapping to genes. The counts were very low for Rbpj (too low for differential expression analysis), but they could have been quite a bit higher if this phantom exon had been included in the annotation.
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Old 07-25-2013, 10:16 PM   #2
Bukowski
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Location: Aberdeen, Scotland

Join Date: Jan 2010
Posts: 388
Default

Quote:
Originally Posted by afkoeppel View Post

I noticed I had a number of reads hit the same location between exons 1 and 2 in all of my samples, but that that region was not annotated as an exon (see attached screen shot).

I checked out the annotations for the gene on ...

UCSC: http://genome.ucsc.edu/cgi-bin/hgTra...ches=NM_009035,

... and Ensembl: http://useast.ensembl.org/Mus_muscul...55779-53657445

In both cases, it appears that there is an exon (or something) between the first and second exons of Rbjp (right where my reads are hitting) in some entries, but not in others.
"Not annotated in IGV" does not mean "Not annotated". IGV's standard gene models are pretty poor for mouse and human (I'm unsure of what the data source is, but they miss lots of exons in genes that are real).

I always load up the Ensembl tracks when looking at RNA-Seq or exome data as the models are somewhat better.

mm9 has been superseded by mm10. On the balance of probability your sequence is right, it is present in some transcripts (as indicated by Ensembl and UCSC) but IGV is not up to date.
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Old 07-30-2013, 05:44 AM   #3
jp.
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Location: NikoNarita.jp

Join Date: Jul 2013
Posts: 142
Question

Hi there
I got error with mouse RNAseq. in cummeRbund, it give me 15000gnes but 0TSS and 0....
I am not sure if it is a cummeRbund problem or cuffdiff ?
can someone help me ?

thank you


Quote:
Originally Posted by Bukowski View Post
"Not annotated in IGV" does not mean "Not annotated". IGV's standard gene models are pretty poor for mouse and human (I'm unsure of what the data source is, but they miss lots of exons in genes that are real).

I always load up the Ensembl tracks when looking at RNA-Seq or exome data as the models are somewhat better.

mm9 has been superseded by mm10. On the balance of probability your sequence is right, it is present in some transcripts (as indicated by Ensembl and UCSC) but IGV is not up to date.
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