Two FPKM values for same gene

Question

[Bioblazer]

CUFFDIFF result

test_id   gene_id   gene    locus   sample_1      sample_2   status value_1 value_2 log2(fold_change)   test_stat   p_value q_value significant

XLOC_009967 XLOC_009967 **AFLA_002470** EQ963482:529554-531870  AT30    AT37    OK  19.4865 13.9912 -0.477954   -0.35927    0.71835 0.999247        no

XLOC_010306 XLOC_010306 **AFLA_002470** EQ963482:529554-531870  AT30    AT37    OK  41.1815 37.0789 -0.151399   -0.213452   0.82825 0.999247       no

I followed nature protocol paper "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks" for RNA -seq Data analysis.

Why cufflinks/cuffdiff generated two locus id for same gene? is it due to alternative splicing site?

Answer 1

[mbk0asis]

They could be transcript variants. Check the GTF file you used.

[Bioblazer]

EQ963482    JCVI    gene    530665  531823  .   -   .   gene_id "CADAFLAG00010313"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding";                                                                                                                     
EQ963482    JCVI    transcript  530665  531823  .   -   .   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding";                                                                                                                        
EQ963482    JCVI    exon    531778  531823  .   -   .   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "1"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; exon_id "CADAFLAE00029154";                                                                                                                       
EQ963482    JCVI    CDS 531778  531823  .   -   0   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "1"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; protein_id "CADAFLAP00010313";                                                                                                                        
EQ963482    JCVI    start_codon 531821  531823  .   -   0   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "1"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding";                                                                                                                       
EQ963482    JCVI    exon    531053  531720  .   -   .   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "2"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; exon_id "CADAFLAE00029155";                                                                                                                       
EQ963482    JCVI    CDS 531053  531720  .   -   2   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "2"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; protein_id "CADAFLAP00010313";                                                                                                                        
EQ963482    JCVI    exon    530665  530967  .   -   .   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "3"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; exon_id "CADAFLAE00029156";                                                                                                                       
EQ963482    JCVI    CDS 530668  530967  .   -   0   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "3"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding"; protein_id "CADAFLAP00010313";                                                                                                                        
EQ963482    JCVI    stop_codon  530665  530667  .   -   0   gene_id "CADAFLAG00010313"; transcript_id "CADAFLAT00010313"; exon_number "3"; gene_name "AFLA_002470"; gene_source "JCVI"; gene_biotype "protein_coding"; transcript_name "AFLA_002470A"; transcript_source "JCVI"; transcript_biotype "protein_coding";

how to know whether gene has transcript variant from gtf file?

[dr_bantz]

yes this does seem likely - although it's the same gene, the XLOC values are different.