Clarification on Tohpat/Cufflinks with Ensembl

[sindrle]

Im currently analysing some mice samples and wanted to try the Ensembl annotation, I have only used UCSC before.

This is my tophat cmd:

tophat2 -o path/to --transcriptome-index=/Mus_musculus_Ensembl_NCBIM37/Mus_musculus/Ensembl/NCBIM37/Annotation/Genes/genes /Mus_musculus_Ensembl_NCBIM37/Mus_musculus/Ensembl/NCBIM37/Sequence/Bowtie2Index/genome 001.fastq.gz

this is my cufflinks cmd:

cufflinks --output-dir path/to --GTF-guide /Mus_musculus_Ensembl_NCBIM37/Mus_musculus/Ensembl/NCBIM37/Annotation/Archives/archive-2013-03-06-18-55-12/Genes/genes.gtf --frag-bias-correct /Mus_musculus_Ensembl_NCBIM37/Mus_musculus/Ensembl/NCBIM37/Sequence/Bowtie2Index/genome.fa --multi-read-correct --upper-quartile-norm --verbose accepted_hits.bam

What worrying?

First:

20: MT
10: 18
11: 19
4: 12
5: 13
2: 10
3: 11
8: 16
9: 17
6: 14
7: 15
22: Y
21: X
19: 9
18: 8
17: 7
16: 6
15: 5
14: 4
13: 3
12: 2
1: 1

Does it seam correct for chromosome names?

Second, maybe 1000 warning like this:

GFF warning: merging adjacent/overlapping segments of ENSMUST00000101224 on 6 (84939386-84939418, 84939421-84939523)

What does that mean?

Third, what the meaning of:

Warning: intron not within scaffold ([102700571-102700733], 0)


Thank you very much!

[Brian Bushnell]

It would be very helpful if you could first explain what kind of input data you have, and what you are trying to accomplish. Also, "path/to" is supposed to be replaced with the path to something - you shouldn't actually specify the literal "path/to".

[sindrle]

It would be very helpful if you could first explain what kind of input data you have, and what you are trying to accomplish. Also, "path/to" is supposed to be replaced with the path to something - you shouldn't actually specify the literal "path/to".

The input data is Illumina HiSeq2000 fastq of mouse tissue.

First, Im trying to align the read to the Ensembl mouse genome, but I want be sure its going well.

Cufflinks will be run on all my samples, followed by Cuffmerge. I want to look at expression levels of all genes, and novel transcripts. But also here I need to be sure everything goes as planned.

The "path/to" is simply there not to reveal my true name etc. which is in the path.

Thank you for quick reply!

[Brian Bushnell]

sindrle,

I am not involved with Bowtie/Tophat/Cufflinks, so I can not offer you direct advice. Hopefully the authors will get back to you!

But if you want to use BBMap, which is faster and more accurate than TopHat, I will be more than happy to help. It produces TopHat-compatible output, with all of the special tags required by Cufflinks.

I'd like to mention, though, that I've recently been dismayed to learn that Cufflinks appears to be incapable of distinguishing between a human male and female using X/Y-specific RNA expression... so I'm looking into alternatives.

[sindrle]

This is with UCSC

20: MT
10: 18
11: 19
4: 12
5: 13
2: 10
3: 11
8: 16
9: 17
6: 14
7: 15
22: Y
21: X
19: 9
18: 8
17: 7
16: 6
15: 5
14: 4
13: 3
12: 2
1: 1


Its the same, so I guess its correct. Confusing name change, but whatever.