Hello All,
I have a drosophila GTF file which I created, since I wanted to use the latest release from flyable and the UCSC annotated GTF is from 2006. Anyway, here is what it looks like…
After running the latest version of Tophat with the one-directional RNA-Seq reads, I get a .bam file. Using this my input to cufflinks was…
After running this using LSF command 'bsub', I get an error output file, the start of which looks like this
I have three output files, the smallest of which is the genes.expr file and the largest is the transcripts.gtf file.
Questions:
1) How will I know if cufflinks has accepted the GTF file that I created correctly or not? I used a perl script from the internet to check the validity of my GTF file. Its output says that barring the non-availability of CDS coordinates the GTF file is ok. Do I need to change the positions of some fields? or add CDS coordinates to the file.
2) Why am I getting such a huge error file? I see that in the UCSD GTF file the chromosome names begin with chr. Do I need to change the annotation to chr3L, chr3R, chr2L, chr2R and so on?
3) When I supply the reference genome, to bowtie-build, I send a single fasta file containing the chromosome names and their sequences. It looks like this.
Do I need to break this up into separate chromosomes/files and build separate indexes? Sounds a bit illogical but I thought I'd ask.
4) I get FPKM values of zero for some genes in the genes.expr file. Do I interpret that as too low expression to call? or is the program not running proper.
Thank you for taking the time to read such a long post. I am relatively new to the cufflinks program, and GTF so any inputs and suggestions would be much appreciated.
Regards
Abhijit
I have a drosophila GTF file which I created, since I wanted to use the latest release from flyable and the UCSC annotated GTF is from 2006. Anyway, here is what it looks like…
Code:
3R FlyBase exon 24574105 24575330 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "7"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24575574 24575753 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "6"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24575893 24576062 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "5"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24576188 24576651 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "4"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24576707 24576885 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "3"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24576947 24577107 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "2"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24577166 24577313 . - . gene_id "FBgn0039596"; gene_name "CG10000"; exon_number "1"; transcript_id "FBtr0085315"; parent_type=mRNA; 3R FlyBase exon 24562831 24563368 . - . gene_id "FBgn0039595"; gene_name "AR-2"; exon_number "4"; transcript_id "FBtr0085316"; parent_type=mRNA; 3R FlyBase exon 24566194 24566352 . - . gene_id "FBgn0039595"; gene_name "AR-2"; exon_number "3"; transcript_id "FBtr0085316"; parent_type=mRNA; 3R FlyBase exon 24566428 24566706 . - . gene_id "FBgn0039595"; gene_name "AR-2"; exon_number "2"; transcript_id "FBtr0085316"; parent_type=mRNA;
Code:
cufflinks -p 4 -G ALLEXONS.gtf -o ./outputdir accepted_hits.bam
Code:
GFF warning: merging adjacent/overlapping segments of FBtr0084817 on 3R (21094383-21094697, 21094700-21095435) [20:04:14] Inspecting reads and determining fragment length distribution. > Processing Locus 2L:21918-25151 [ ] 0% > Processing Locus 2L:76445-77211 [ ] 0% > Processing Locus 2L:102381-106718 [ ] 0%
Questions:
1) How will I know if cufflinks has accepted the GTF file that I created correctly or not? I used a perl script from the internet to check the validity of my GTF file. Its output says that barring the non-availability of CDS coordinates the GTF file is ok. Do I need to change the positions of some fields? or add CDS coordinates to the file.
2) Why am I getting such a huge error file? I see that in the UCSD GTF file the chromosome names begin with chr. Do I need to change the annotation to chr3L, chr3R, chr2L, chr2R and so on?
3) When I supply the reference genome, to bowtie-build, I send a single fasta file containing the chromosome names and their sequences. It looks like this.
Code:
>Y AGCTAGCT >2L GCTGCTGCAGTC >2R CGATGATGA
4) I get FPKM values of zero for some genes in the genes.expr file. Do I interpret that as too low expression to call? or is the program not running proper.
Thank you for taking the time to read such a long post. I am relatively new to the cufflinks program, and GTF so any inputs and suggestions would be much appreciated.
Regards
Abhijit