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)