Cufflinks not masking features in the mask GTF file?

[rflrob]

Hi all,
I'm running cufflinks with the mask option:

   cufflinks --num-threads 50 --output-dir analysis-multi/CaS3A -u -M Reference/AAA/suspect.gtf  --frag-bias-correct Reference/AAA/mel_only.fa  -G Reference/AAA/mel_only.gtf analysis-multi/CaS3A/assigned_dmel_rescued.bam

but I still end up with, for instance, relatively high (and variable) FPKMs in mitochondrial genes, which should be filtered out from the reads.

$ grep mt:tRNA:K mel_only.gtf
dmel_dmel_mitochondrion_genome	FlyBase	exon	3768	3838	.	+	.	transcript_id "FBtr0100864"; gene_id "FBgn0013697"; gene_name "mt:tRNA:K";
$ grep mt:tRNA:K suspect.gtf
dmel_dmel_mitochondrion_genome	FlyBase	exon	3768	3838	.	+	.	transcript_id "FBtr0100864"; gene_id "FBgn0013697"; gene_name "mt:tRNA:K";

I did find an old, essentially un-answered post on this forum mentioning a similar issue; anyone have any other deep insight?

[pengchy]

I have compared the speed of running with " -M -g " parameters and without them.
The "without" group only lasted 3-5 hours, but the "with" group have been running 48 hours without any output.

[sschavan]

cufflinks masking option for mitochondrial genes

Hi all,

I have a similar problem, I tried using the cufflinks masking option for mitochondrial genes using the below command:

cufflinks --mask-file=/illumina/genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/mask_MR.gtf -o . -p 6 -g /illumina/genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf -u ../tophat_HS034/accepted_hits.bam

In the resultant cufflinks output file, genes.fpkm_tracking and isoforms.fpkm_tracking I see that the MT were still present so I couldn't get the masking option to work

I also tried both -g / -G , didn't work for them too. I wasn't sure if I am doing something incorrectly.

I was wondering if there is anyone who could get masking option to work and could provide any guidance, that would be highly appreciated.

Thanks
Shweta

[sindrle]

Can I ask how you created this file?

/illumina/genomes/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/mask_MR.gtf

[sschavan]

I just 'awk' the lines having Chromosome column value as "MT" from the genes.gtf file to get a subset of mitochondrial entries. I obtained a subset from the Ensembl gtf file, which had "MT" as Chromosome in the column1.

awk -F "\t" '$1=="MT"{print }' < genes.gtf > mask_MR.gtf

Shweta