FASTX Toolkit barcode splitter issue

[upendra_35]

Hi jdanderson,
I think your command looks good to me and i suspect the problem is with the barcode file.Try opening the barcode file with vi and see if there is anything werid going on. Sometimes you see ^M at the end of the line and if you see so then you can manually fix this and re-run the command. Good luck....

[2007lab]

Bump for the solid part of this thread.
Once I run the solid2fastq.pl to convert my csfasta and qual to a fastq.gz file, can I use fastx to do QC on my solid PE reads?

[jdanderson]

Hello Kevin,

I am not sure. I cannot directly tell from the documentation, however, i don't see any mention of color space reads. Maybe you could query the Hannon Lab if you don't get an immediate answer on here ([email protected]).

-
Johnathon

[hyjkim]

Fastx toolkit does not work for solid data. I wrote some perl scripts to demultiplex some solid data few months back. The code and the syntax weren't pretty. If you're interested, I can dig the scripts up and post them.

[KevinLam]

sorry to hijack your thread but would fastx toolkit be able to demultiplex SOLiD reads as well?

[jdanderson]

Hello All,

I am updating my progress in case this may help someone in the future.

As previously mentioned I used the FASTX Toolkit on the export.txt and extended.txt files from Illumina pipeline 1.6 with minimal success and I suspected a formatting error in these files. I just tried using the same Barcode Splitting module on the sequence.txt file (prior to reformatting to Sanger Fastq) and it seems to have worked fine, with the caveat that there appears to be more reads in the unmatched file than I had expected (199,524 out of 28,223,602 or 0.7%), but perhaps this is normal. For reference, I had used the NuGen Ovation and Encore Kits for library prep.

Regards,
Johnathon

[jdanderson]

Hello All,

I've been trying to use the FASTX Toolkit barcode splitter to demultiplex my illumina reads. The following command runs okay without any errors:

[cat /home/johnathon/jda_ev_extended.txt | fastx_barcode_splitter.pl --bcfile /home/johnathon/mybarcodes.txt --bol --mismatches 1 --prefix /home/johnathon/split_bc/jda_ev_split_bc --suffix ".txt"]

But none of the output files contain any reads except for the mismatched file.

The following is mybarcode.txt file:

[#I hope the following is the appropriate format for this txt file, it should contain the barcode identifier and the barcode sequence itself in a tab delimited fashion--Johnathon David Anderson
BC1 ACCC
BC2 CGTA
BC3 GAGT
BC4 TTAG]


However, when I look at the extended.txt file i can see the right barcodes on the 5' end. I have also tried to use the export.txt file to no avail; apparently it is not formatted appropriately. I get an error message saying for the first character there is an "S" instead of an "<" or an "@".

I have not converted these files from Solexa to Sanger Fastq. Could this be the issue?

For my first data set that was not barcoded I was using the MAQ fq_all2std.pl script export2std command to convert the export.txt file. It worked just fine and I was able to visualize the data on IGV. I haven't had much success with MAQ patch ill2sanger and am wondering if this is the issue with FASTX Toolkit, then can anyone recommend a user friendly script to convert. I am using Solexa pipeline 1.6.

Is anyone familiar with the FASTX Toolkit? Is the problem probably that the Illumina files need to be converted to Sanger FASTQ first?

Any guidance would be most appreciated?

Regards,
Johnathon