Hello,

It looks like the data is mapping as novel - not linked with the reference annotation. There can be a few factors that can cause this to occur for part of a dataset (often desirable) but when it occurs for an entire dataset, there is often a data mismatch or parameter issue.


The first item I always check is that the reference genomes are a match between inputs. Do this by confirming that the identifiers in the reference GFF file are the same as those in the Tophat BAM output (convert to SAM, with headers, to see the chromosome names). For the GFF file, the tool " Join, Subtract and Group -> Group" on the first column, chromosome name, with the action "count distinct" will isolate these.

But the real problem could be in the parameters, see below:

On 1/11/14 10:43 PM, Yang Bi wrote:
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).
My guess is that this Cufflinks run had the same issue - have you checked it? The 'Min isoform fraction' set to "0" may be problematic (I have never run Cufflinks this way). It may seem that this is a setting that is permissive - to capture even very small expression levels - but it may have had the reverse effect of not assigning any reads.

(The Tophat run with min intron at 20 is pretty low/sensitive - but with a smaller genome this probably will not cause memory issues with the mapping. Was this set based on the genome having transcripts with known, characterized introns this short? I didn't check, but you can in the reference GFF file.).

Maybe double check the above Cufflinks run, confirm the results were as expected, then try the default in Cufflinks to see how that works out ("0.1")? As a first pass test? If you want to make this more sensitive in subsequent run, you could try "0.01" - although how significant those results are, given this genome and your specific input data, would need to be evaluated.

After that, if you are still having trouble, please feel free to share a history link and we can try to help (copy and email a share link from the public server, direct to me, to keep your data private). Here is how:
https://wiki.galaxyproject.org/Support#Shared_and_Published_data

Hopefully the parameter change works, or a reference genome issue is found and corrected, but if not, I'll watch for your email,

Jen
Galaxy team

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
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
using "reply all" in your mail client.  For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

   http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists,
please use the interface at:

   http://lists.bx.psu.edu/

To search Galaxy mailing lists use the unified search at:

   http://galaxyproject.org/search/mailinglists/

--
Jennifer Hillman-Jackson
http://galaxyproject.org

___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
using "reply all" in your mail client.  For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

  http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists,
please use the interface at:

  http://lists.bx.psu.edu/

To search Galaxy mailing lists use the unified search at:

  http://galaxyproject.org/search/mailinglists/

Reply via email to