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
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