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 <veranj...@yahoo.com>
To: galaxy-user <galaxy-user@lists.bx.psu.edu>
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
Veranja Liyanapathirana
Graduate Student (Microbiology)
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