Trying to make sense of cufflinks output (--GTF vs. --GTF-guide)

lethalfang

Member

Join Date: Aug 2011
Posts: 95

Trying to make sense of cufflinks output (--GTF vs. --GTF-guide)

05-25-2014, 12:27 PM

Hi guys,

This isn't the first time this question is posted on this board, but I haven't found an answer yet.

I tested out Cufflinks on a TCGA data set, using both -GTF and -GTF-guide optinos:

Code:

# --GTF option
cufflinks -o Test01G -p 4 -G genes.gtf -u --library-type fr-unstranded TCGA.bam

# --GFT-guide option
cufflinks -o Test11g -p 4 -g genes.gtf -u --library-type fr-unstranded TCGA.bam

Then, I looked for the results for some "house keeping" genes used in qPCR such as GAPDH and ACTB. Let's look at the results.

First, --GTF option:

When I grep for 'ACTB|GAPDH' in the isoforms.fpkm_tracking file:

Code:

tracking_id	class_code	nearest_ref_id	gene_id	gene_short_name	tss_id	locus	length	coverage	FPKM	FPKM_conf_lo	FPKM_conf_hi	FPKM_status
NM_002046	-	-	GAPDH	GAPDH	TSS28915	chr12:6643584-6647537	1383	181.604	95.7623	90.8633	100.661	OK
NM_001256799	-	-	GAPDH	GAPDH	TSS12547	chr12:6644408-6647537	1407	0.432855	0.22825	0.0308549	0.425646	OK
NM_001101	-	-	ACTB	ACTB	TSS29883	chr7:5566778-5570232	1812	694.784	366.533	361.681	371.386	OK

When I grep for 'ACTB|GAPDH' in the transcripts.gtf file:

Code:

chr12	Cufflinks	transcript	6643585	6647537	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6643585	6643735	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "1"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6643976	6644027	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "2"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6645660	6645759	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "3"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6645850	6645956	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "4"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6646086	6646176	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "5"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6646267	6646382	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "6"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6646475	6646556	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "7"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6646750	6647162	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "8"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	exon	6647267	6647537	1000	+	.	gene_id "GAPDH"; transcript_id "NM_002046"; exon_number "9"; FPKM "95.7623252884"; frac "0.997574"; conf_lo "90.863255"; conf_hi "100.661396"; cov "181.604009";
chr12	Cufflinks	transcript	6644409	6647537	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6644409	6644635	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "1"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6645660	6645759	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "2"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6645850	6645956	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "3"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6646086	6646176	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "4"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6646267	6646382	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "5"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6646475	6646556	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "6"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6646750	6647162	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "7"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr12	Cufflinks	exon	6647267	6647537	2	+	.	gene_id "GAPDH"; transcript_id "NM_001256799"; exon_number "8"; FPKM "0.2282504343"; frac "0.002426"; conf_lo "0.030855"; conf_hi "0.425646"; cov "0.432855";
chr7	Cufflinks	transcript	5566779	5570232	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5566779	5567522	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "1"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5567635	5567816	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "2"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5567912	5568350	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "3"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5568792	5569031	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "4"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5569166	5569294	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "5"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";
chr7	Cufflinks	exon	5570155	5570232	1000	-	.	gene_id "ACTB"; transcript_id "NM_001101"; exon_number "6"; FPKM "366.5331600779"; frac "1.000000"; conf_lo "361.680624"; conf_hi "371.385696"; cov "694.783972";

I think those results are quite reasonable.

Then, I looked at the results for the --GTF-guide option:
When I grep for 'ACTB|GAPDH' in the isoforms.fpkm_tracking file:

Code:

NM_002046	-	-	GAPDH	-	-	chr12:6643584-6647537	1383	182.046	95.9885	91.0819	100.895	OK
NM_001256799	-	-	GAPDH	-	-	chr12:6644408-6647537	1407	0	0	0	0.0420819	OK

There is no ACTB, but when I looked for the coordinates:

Code:

NM_001101	-	-	CUFF.10954	-	-	chr7:5566778-5570232	1812	0	0	0	0.0314856	OK

Why isn't there any sign of ACTB using the --GTF-guide option? ACTB is typically used as a house keeping gene in qPCR, so I figure it should be there.

Also, ACTB does not appear in the transcripts.gtf file, either.

Can someone explain the differences between --GTF and --GTF-guide?

Thanks in advance.

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