I am new to RNAseq and before run my counts I am looking at my files on IGV to look at coverage of some specific genes as a quick QC. I am doing my work in Arabidopsis and I appear to have no reads mapping to the plastid (chloroplast) genes. All 5 of the other chromosomes and the mitochondrial genome are present but not the plastid. This surprises me because during my QC before alignment I saw that one of my over-represented sequences was a chloroplast gene. Does anyone know what the problem could be?
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Most likely the reference genome used during alignment did not include the chloroplast sequence.Originally posted by JustinH View Post
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Phillip -
Was the overrepresented sequence nuclear encoded?
And did you do polyA enriched RNA and are wondering about the lack of plastome encoded gene expression?
As polyadenylation has a very different meaning in chloroplasts.
In any case maybe this review
http://www.plantphysiol.org/content/155/4/1520.full
can help you if you talk about plastid encoded genes.
Cheers
BjörnComment
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The over-represented sequence was PsbA which is chloroplast encoded. We did not do a poly-a enrichment to avoid loosing chloroplast encoded genes but we did do the RNA depletion. From what I can tell the reference genome did contain the Plastid genome in both the genome and annotation file. Thanks,
JustinComment
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Is it possible that the name of the plastid sequence was different between the plastid sequence used as a reference for alignment and the plastid sequence used for the IGV .genome file?Originally posted by JustinH View Post
Because typically .bam files do not have the reference sequence to which the reads in question are aligned embedded within it, that sequence must be obtained from somewhere else during display within a viewer. IGV uses .genome files.
However, if there are sequences within the .genome with names that don't match the .bam file reference names, then IGV will not display them.
Do you have a way to run samtools on the .bam file? If you could post the results of:
samtools idxstats yourbamfile.bam
(where you replace "yourbamfil.bam" with the name of your particular bam file) we could troubleshoot this more effectively.
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PhillipComment
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Phiilip,
Thanks for that advice. I ran the code and got the input below which I interpreted as my sample having plastid genes, and something in the IGV reference genome wasn't finding them. I then learned I can use my own genome file and loaded the one I have been using and saw the plastid reads. Thank you so much that was relief to see.
Justin
1 30427671 3776614 0
2 19698289 3406598 0
3 23459830 2052135 0
4 18585056 1630542 0
5 26975502 2348291 0
Mt 366924 426293 0
Pt 154478 11432972 0
* 0 0 0Comment
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Sure, glad that worked out for you. Actually, I knew there was no way you could get an RNA data set from a plant without hits in the plastid genome.Originally posted by JustinH View Post
This is a perennial issue with IGV. Or with .bam files, depending on your vantage point.
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