Dear all,
I was using the barcode splitter on Miseq paired end reads, however I am not 
sure if I did it correctly as the results I get in terms of the number of reads 
alocated per each barcode does not tally with the resutls obtained by the our 
service provider by one of their in-house script based methods. I use it for 
splitting some inhouse barcodes. I need to make sure that read 1 and read 2 are 
split in to the same group, and drop the sequences where this criteria is not 
met. Not sure how to get about doing this. Would using FASTQ joiner on the two 
reads and subsequent splitting work? 
Thank you,
Kind Regards,

 From: Veranja Liyanapathirana <>
To: galaxy-user <> 
Sent: Saturday, 6 April 2013, 23:13
Subject: Error in creating Depth of Coverage files after  Bowtie for Illumina 

Dear Galaxy team/ users,
I am sorry to spam the thread again but I still could not figure out what is 
worng with my work flow and need some help. 
As mentioned earlier, I use Miseq reads, demultiplex for an inhouse barcode 
using barcode splitter, re-upload and map with a ref sequence that is 
consisting of multiple short reference sequences. The work flow goes well up to 
this stage, conversion from SAM to BAM  after filtering the SAM files also fine 
but I can not use the GATK depth of coverage tool to get the alignment data or 
create pileups. An error comes up in all instances. 
I would really appreciate any inputs in to this.
Thanks a lot,
Veranja Liyanapathirana
Graduate Student (Microbiology) 

 From: Veranja Liyanapathirana <>
To: galaxy-user <> 
Sent: Thursday, 4 April 2013, 6:39
Subject: Using segments of sequences as a reference genome - Bowtie for Illumina

Dear all, 
My problem seems like something that should have a very simple solution from my 
end and due to my lack of knowledge in bioinformatics, I am probably messing up 
with the workflows. The experiment I run is one where we used Miseq to sequence 
amplicons of a multiplex PCR. We introduced an inhouse barcodeto our PCR 
products via an adaptor.  

Miseq data was demultiplexed for the Illumina barcodes using Miseq reporter on 
intrument software by our service provider and I am trying to run the rest of 
the process on Galaxy web port with no command prompt programming.   

The data for R1 and R2 was imported, and then I used barcode splitter to 
de-multiplex the amplicons after quality triming. (I did not use FASTQ groomer 
as Miseq data is supposed to be Sanger FastQ than Illumina).  

Then the sequence trimmer was used to trim the barcode+adaptor sequences. The 
results of this were re-uploaded and designated as FASTQ for alignment. 

Now for the reference genome, as our aplicons are of from different sequences, 
we have segmented FASTA sequences in one file with different FASTA identifiers. 
When this file was input as the reference genome and mapping was performed 
using Bowtie for Illumina, the mapping went on with no errors.  

I could filter the alignment file using SAM filters too. But I can not do any 
more downstream visualozations, not even  SAM to BAM conversion.  

I suspect that this may be due to an error in the way that the reference genome 
was formulated but can not get around to figure it out. I would be extremely 
grateful if you could help me with this issue. I tihnk if I string together the 
sequences as one it would work, but converting this back for interpretation 
becomes an issue then. 

Thank you,
Kind Regards,
Veranja Liyanapathirana
Graduate Student (Microbiology)
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at  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:

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

To search Galaxy mailing lists use the unified search at:

Reply via email to