To all Cufflinks users and author, can anyone please comment if I have stated my problem clearly.
Unfortunately it may be a serious bug in this software, which is causing it to mix FPKMs between different genes. Not only it obviously makes cufflinks highly unreliable, but a puts a question mark over the published findings.
Thanks for your attention and replies.
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Cufflinks annotation handling business
Help me understand if you know, or comment if you find similar
Cufflinks run with -g, using GTF (from illumina igenomes).
ORIGINAL ANNOTATION
Code:chr2 unknown exon 152163161 152164423 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown stop_codon 152163943 152163945 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown CDS 152163946 152164423 . - 1 gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown CDS 152165451 152165743 . - 0 gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown exon 152165451 152165743 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown CDS 152168773 152169063 . - 0 gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown exon 152168773 152169063 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown start_codon 152169061 152169063 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; chr2 unknown exon 152169593 152169796 . - . gene_id "Trib3"; gene_name "Trib3"; p_id "P4503"; transcript_id "NM_175093"; tss_id "TSS19853"; ;
CUFFLINKS OUTPUT:
What happened to gene ID?? where did it go
Code:chr2 Cufflinks transcript 152163161 152169796 1000 - . gene_id "CUFF.9451"; transcript_id "NM_175093"; FPKM "35.0692808303"; frac "0.811271"; conf_lo "33.828739"; conf_hi "36.309823"; cov "154.402273"; full_read_support "yes"; chr2 Cufflinks exon 152163161 152164423 1000 - . gene_id "CUFF.9451"; transcript_id "NM_175093"; exon_number "1"; FPKM "35.0692808303"; frac "0.811271"; conf_lo "33.828739"; conf_hi "36.309823"; cov "154.402273"; chr2 Cufflinks exon 152165451 152165743 1000 - . gene_id "CUFF.9451"; transcript_id "NM_175093"; exon_number "2"; FPKM "35.0692808303"; frac "0.811271"; conf_lo "33.828739"; conf_hi "36.309823"; cov "154.402273"; chr2 Cufflinks exon 152168773 152169063 1000 - . gene_id "CUFF.9451"; transcript_id "NM_175093"; exon_number "3"; FPKM "35.0692808303"; frac "0.811271"; conf_lo "33.828739"; conf_hi "36.309823"; cov "154.402273"; chr2 Cufflinks exon 152169593 152169796 1000 - . gene_id "CUFF.9451"; transcript_id "NM_175093"; exon_number "4"; FPKM "35.0692808303"; frac "0.811271"; conf_lo "33.828739"; conf_hi "36.309823"; cov "154.402273";
CUFFMERGE OUTPUT:
ok, got my gene name back!
What about gene_id? What happened to transcript ID? Where did CUFF.7092.2 come from? Where happened to p_ID?
Code:chr2 Cufflinks exon 152163157 152164423 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023065"; exon_number "1"; gene_name "Trib3"; oId "CUFF.7092.2"; nearest_ref "NM_175093"; class_code "j"; tss_id "TSS16814"; chr2 Cufflinks exon 152165420 152165427 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023065"; exon_number "2"; gene_name "Trib3"; oId "CUFF.7092.2"; nearest_ref "NM_175093"; class_code "j"; tss_id "TSS16814"; chr2 Cufflinks exon 152163157 152164423 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023066"; exon_number "1"; gene_name "Trib3"; oId "CUFF.7092.1"; nearest_ref "NM_175093"; class_code "j"; tss_id "TSS16815"; chr2 Cufflinks exon 152166010 152166019 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023066"; exon_number "2"; gene_name "Trib3"; oId "CUFF.7092.1"; nearest_ref "NM_175093"; class_code "j"; tss_id "TSS16815"; chr2 Cufflinks exon 152163161 152164423 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023067"; exon_number "1"; gene_name "Trib3"; oId "NM_175093"; nearest_ref "NM_175093"; class_code "="; tss_id "TSS16816"; p_id "P12953"; chr2 Cufflinks exon 152165451 152165743 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023067"; exon_number "2"; gene_name "Trib3"; oId "NM_175093"; nearest_ref "NM_175093"; class_code "="; tss_id "TSS16816"; p_id "P12953"; chr2 Cufflinks exon 152168773 152169063 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023067"; exon_number "3"; gene_name "Trib3"; oId "NM_175093"; nearest_ref "NM_175093"; class_code "="; tss_id "TSS16816"; p_id "P12953"; chr2 Cufflinks exon 152169593 152169796 . - . gene_id "XLOC_013512"; transcript_id "TCONS_00023067"; exon_number "4"; gene_name "Trib3"; oId "NM_175093"; nearest_ref "NM_175093"; class_code "="; tss_id "TSS16816"; p_id "P12953";
CUFFDIFF OUTPUT:
I will be damned!
Whats with the new TSS ID. IN my original GTF, this ID belongs to an entirely different gene (LOC100040911) on chrY. For which gene am i looking at expression difference -- Trib3 or LOC100040911?
Code:tracking_id class_code nearest_ref_id gene_id gene_short_name tss_id locus length coverage q1_FPKM q1_conf_lo q1_conf_hi q1_status q2_FPKM q2_conf_lo q2_conf_hi q2_status TSS16815 XLOC_013512 Trib3 chr2:152163156-152169796 q1 q2 OK 0.00575202 14.359 11.2856 -2.70945 0.0067394 0.0480599 yes
Last edited by epi; 03-01-2012, 08:00 AM.
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