Dear all: I am new to Galaxy and I followed online tutorials/tips to analyze my RNA seq data for alternative splicing. I used "tophat for illumina" to align my sequencing data after QC/filtering. Other than setting min intron to 20, I used the default settings. Then I feed the accepted hit files to cufflink. I set Min isoform fraction to 0, use annotation (tair10 gff3) as guide and choose yes for perform bias correction (locally cached tair10). I merged the assembled transcripts with cuffmerge and use cuffcompare to compare the resultant merged assembled transcript to the reference annotation file tair10 gff3. I choose yes for "use sequence data" and locally cached tair10 as the "reference list". I get this for the transcript accuracy analysis: # Cuffcompare v2.1.1 | Command line was: #cuffcompare -o cc_output -r /galaxy-repl/main/files/007/386/dataset_7386886.dat -s /galaxy/data/Arabidopsis_thaliana_TAIR10/sam_index/Arabidopsis_thaliana_TAIR10.fa ./input1 # #= Summary for dataset: ./input1 : # Query mRNAs : 72778 in 51779 loci (57559 multi-exon transcripts) # (12679 multi-transcript loci, ~1.4 transcripts per locus) # Reference mRNAs : 42163 in 33350 loci (30127 multi-exon) # Corresponding super-loci: 33140 #--------------------| Sn | Sp | fSn | fSp Base level: 100.0 62.7 - - Exon level: 104.6 59.5 100.0 60.5 Intron level: 100.0 55.5 100.0 56.5 Intron chain level: 98.3 51.5 100.0 60.3 Transcript level: 98.7 57.2 94.8 54.9 Locus level: 99.4 64.0 100.0 64.1 Matching intron chains: 29618 Matching loci: 33147 Missed exons: 1/169820 ( 0.0%) Novel exons: 128021/298149 ( 42.9%) Missed introns: 0/127896 ( 0.0%) Novel introns: 102614/230568 ( 44.5%) Missed loci: 1/33350 ( 0.0%) Novel loci: 2962/51779 ( 5.7%) Total union super-loci across all input datasets: 51779 For the tmap file, all my FPKMs are 0: ref_gene_id ref_id class_code cuff_gene_id cuff_id FMI FPKM FPKM_conf_lo FPKM_conf_hi cov len major_iso_id ref_match_len AT1G01010 AT1G01010.1 = AT1G01010 TCONS_00000001 0 0.000000 0.000000 0.000000 0.000000 1688 TCONS_00000001 1688 AT1G01040 AT1G01040.1 = AT1G01040 TCONS_00000002 0 0.000000 0.000000 0.000000 0.000000 6251 TCONS_00000002 6251 AT1G01040 AT1G01040.2 = AT1G01040 TCONS_00000003 0 0.000000 0.000000 0.000000 0.000000 5877 TCONS_00000002 5877 AT1G01046 AT1G01046.1 = AT1G01046 TCONS_00000004 0 0.000000 0.000000 0.000000 0.000000 207 TCONS_00000004 207 AT1G01073 AT1G01073.1 = AT1G01073 TCONS_00000005 0 0.000000 0.000000 0.000000 0.000000 111 TCONS_00000005 111 AT1G01110 AT1G01110.2 = AT1G01110 TCONS_00000006 0 0.000000 0.000000 0.000000 0.000000 1782 TCONS_00000006 1782 AT1G01110 AT1G01110.1 = AT1G01110 TCONS_00000007 0 0.000000 0.000000 0.000000 0.000000 1439 TCONS_00000006 1439 AT1G01115 AT1G01115.1 = AT1G01115 TCONS_00000008 0 0.000000 0.000000 0.000000 0.000000 117 TCONS_00000008 117 AT1G01160 AT1G01160.1 = AT1G01160 TCONS_00000009 0 0.000000 0.000000 0.000000 0.000000 1045 TCONS_00000010 1045 AT1G01160 AT1G01160.2 = AT1G01160 TCONS_00000010 0 0.000000 0.000000 0.000000 0.000000 1129 TCONS_00000010 1129 AT1G01180 AT1G01180.1 = AT1G01180 TCONS_00000011 0 0.000000 0.000000 0.000000 0.000000 1176 TCONS_00000011 1176 AT1G01210 AT1G01210.1 = AT1G01210 TCONS_00000012 0 0.000000 0.000000 0.000000 0.000000 616 TCONS_00000012 616 AT1G01220 AT1G01220.1 = AT1G01220 TCONS_00000013 0 0.000000 0.000000 0.000000 0.000000 3532 TCONS_00000013 3532 The FPKMs were normal in the assembled trancripts produced by cufflink. Please enlighten me on the possible mistakes that i have made. I really appreciate your help. Best Yang