An odd error message from Tophat

[Mark.hz]

Hi all,
I am running Tophat/Cufflinks on a RNA-seq datasets(9 samples) from ABI SOLiD. When went through Tophat(v1.1.2), only one sample was terminated with an error message, the rest were doing well and results have already been there.
So it is pretty strange only one sample encountered this error:

[Tue Nov 23 15:14:39 2010] Beginning TopHat run (v1.1.2)
-----------------------------------------------
[Tue Nov 23 15:14:39 2010] Preparing output location tophat_SN94.sam.cfasta/
[Tue Nov 23 15:14:39 2010] Checking for Bowtie index files
[Tue Nov 23 15:14:39 2010] Checking for reference FASTA file
[Tue Nov 23 15:14:39 2010] Checking for Bowtie
Bowtie version: 0.12.7.0
[Tue Nov 23 15:14:39 2010] Checking for Samtools
Samtools version: 0.1.7.0
[Tue Nov 23 15:15:23 2010] Checking reads
min read length: 50bp, max read length: 50bp
format: fasta
[Tue Nov 23 15:30:24 2010] Reading known junctions from GFF file
[Tue Nov 23 15:38:10 2010] Mapping reads against hg19 with Bowtie
[Wed Nov 24 00:51:24 2010] Joining segment hits
Traceback (most recent call last):
File "/usr/local/bin/tophat", line 2201, in <module>
sys.exit(main())
File "/usr/local/bin/tophat", line 2160, in main
user_supplied_juncs)
File "/usr/local/bin/tophat", line 1870, in spliced_alignment
segment_len)
File "/usr/local/bin/tophat", line 1593, in split_reads
split_record(read_name, read_seq, read_quals, output_files, offsets, color)
File "/usr/local/bin/tophat", line 1526, in split_record
read_seq_temp = convert_color_to_bp(read_seq)
File "/usr/local/bin/tophat", line 1500, in convert_color_to_bp
base = decode_dic[base+ch]
KeyError: 'CN'

Has anyone else seen an error like this? Any ideas on what is the reason?
Thanks!

[havard]

This has been discussed in previous threads, for example;

http://seqanswers.com/forums/showthread.php?p=26692

The new versions of TopHat should have been fixed, but I encountered the same problem as you. dcjones wrote in the thread linked above;

I don't thing there is a problem with the '.'s needing to be 'N's. It expects '.'s in colorspace reads. The problem is that tophat converts the '.'s to 'N's on exactly one read (the last read), and it should not.

I don't know that you can modify your reads to work around that.

In my dataset the last 80-90 reads contained '.' , so I removed these. When last read in the dataset did not contain a '.' it seems to work. Maybe the sample that crashed is the only one containg a '.' in the last read?

Håvard

[Mark.hz]

Hi, Havard. Thank you for the suggestion! I've trimmed off the last 600 reads contained ".", and it is running well now. Hope these removed reads have no effect on the result.

[Daehwan]

I'll fix this problem in the next release of TopHat.

[yeyeming]

I'll fix this problem in the next release of TopHat.

hello,Daehwan,do you mean you have fixed it in the new version tophat-1.2.0? but the same error comes when i run it.I do want to know what attribute to such problem

[Daehwan]

hello,Daehwan,do you mean you have fixed it in the new version tophat-1.2.0? but the same error comes when i run it.I do want to know what attribute to such problem

Hi,

We haven't released TopHat 1.2.0, yet.. So I guess I gave you some temporary version by email, which didn't include the fix.

For the time being, you might want to change tophat (or tophat.py) a little bit, change "decode_dic" by the following.
decode_dic = { 'A0':'A', 'A1':'C', 'A2':'G', 'A3':'T', 'A4':'N', 'A.':'N', 'AN':'N',
'C0':'C', 'C1':'A', 'C2':'T', 'C3':'G', 'C4':'N', 'C.':'N', 'CN':'N',
'G0':'G', 'G1':'T', 'G2':'A', 'G3':'C', 'G4':'N', 'G.':'N', 'GN':'N',
'T0':'T', 'T1':'G', 'T2':'C', 'T3':'A', 'T4':'N', 'T.':'N', 'TN':'N',
'N0':'N', 'N1':'N', 'N2':'N', 'N3':'N', 'N4':'N', 'N.':'N', 'NN':'N',
'.0':'N', '.1':'N', '.2':'N', '.3':'N', '.4':'N', '..':'N', '.N':'N' }

[yeyeming]

Hi,

We haven't released TopHat 1.2.0, yet.. So I guess I gave you some temporary version by email, which didn't include the fix.

For the time being, you might want to change tophat (or tophat.py) a little bit, change "decode_dic" by the following.
decode_dic = { 'A0':'A', 'A1':'C', 'A2':'G', 'A3':'T', 'A4':'N', 'A.':'N', 'AN':'N',
'C0':'C', 'C1':'A', 'C2':'T', 'C3':'G', 'C4':'N', 'C.':'N', 'CN':'N',
'G0':'G', 'G1':'T', 'G2':'A', 'G3':'C', 'G4':'N', 'G.':'N', 'GN':'N',
'T0':'T', 'T1':'G', 'T2':'C', 'T3':'A', 'T4':'N', 'T.':'N', 'TN':'N',
'N0':'N', 'N1':'N', 'N2':'N', 'N3':'N', 'N4':'N', 'N.':'N', 'NN':'N',
'.0':'N', '.1':'N', '.2':'N', '.3':'N', '.4':'N', '..':'N', '.N':'N' }

Hello,Daehwan,many thanks ,your suggestion fixed the problem.