Dear Dr. Jennifer,
 
Thank you very much for the reply. I am sorry about the way the thread was 
handled. Your input is much helpful. However, I have one key question which 
probably didnt come across well in the third question and I will try to re-word 
it.
I am using Illumina paired end data and need to de-multiplex some inhouse 
barcodes. I would like to know how best to use "barcode splitter" for this. 
I can think of two ways,
1. to use on read 1 and two separately
2. To join read 1 and read 2 via FASTQ joiner and use the barcode splitter on 
the joined data - use FASTQ splitter prior to mapping.
 
In method one, I need to figure out a way to exclude reads that are not sorted 
in to the same split group in read one and two and discard them from subsequent 
analysis, the possible way to do this as far as I can figure out seems to be 
again to use FASTQ joiner so that the reads without the same identifier in R1 
and R2 would be discarded. Is there any other ways to do this? 
Also, when using the barcode files for read one and read two, is there a need 
to change the "orientation" (i.e complement) of the barcode in the barcode file 
for read 2? 
 
In using the second method, when one uses the barcode splitter, would the 
barcode splitter look at both R1 and R2 or just the R1 in splitting the reads?
 
Thanks a lot,
Kind Regards,
Veranja 
 
 
 
 

________________________________
 From: Jennifer Jackson <j...@bx.psu.edu>
To: Veranja Liyanapathirana <veranj...@yahoo.com> 
Cc: galaxy-user <galaxy-user@lists.bx.psu.edu> 
Sent: Wednesday, 10 April 2013, 5:20
Subject: Re: [galaxy-user] Barcode splitter on paired end data
  

Hi Veranja,

I am going to try to address all questions in one go since they are
    all in the same thread. Next time though, it would be best send new
    questions as a brand new question, not as a reply with just the
    subject line changed. This helps us greatly with tracking and other
    users when searching prior posts.

In the first email you seemed to have some trouble with the format
    of your custom reference genome, but later in the second email this
    seems to be resolved, at least as far as format is concerned
    (SAM->BAM conversion is possible using this genome, in Galaxy?).
    I am going to point you to our help for custom reference genomes,
    and if you click through to the main page there is a table with
    detailed format troubleshooting help. But, I will tell you first
    that I do not believe that this is going to be helpful for your
    overall goals, if I am understanding correctly.

But, here is the link:
http://wiki.galaxyproject.org/Support#Custom_reference_genome

Your reference genome sounds as if it is not really a reference
    genome but instead more of a collection of short read sequences? If
    this number is very large, and the sequences are very short, you
    will likely run into memory or related indexing problems with many
    tools. There really isn't an easy way around this. You could try
    taking the analysis to a cloud version of Galaxy and scaling up the
    memory to see if that helps. You also might try breaking the job up
    into smaller jobs - you mentioned that the data is from multiple
    genomes - perhaps split by genome. But you will have to test this -
    I don't know the actual profile of your data. I can let you know
    that using purely a short read dataset, in particular one that has
    redundancy, will be problematic, likely no matter what is attempted.
    Some assembly or other strategy is likely required to move forward.

Galaxy CloudMan:
http://usegalaxy.org/cloud

For the last question, different tools are probably expected to vary
    a bit in the results since they use a different method. If you want
    to compare datasets, using identifiers would be a good way. Convert
    the files to tabular, cut out the identifiers, compare these to find
    differences, then adjust the tabular files as needed, and convert
    back to fastq/fasta. Tools to do these sorts of functions are in the
    tool groups "Text Manipulation",
    
    "FASTA manipulation", "Filter and Sort, and Join", "Subtract and
    Group", "NGS: QC and manipulation". I know that seems like a lot of
    places to look - but use the tool search at the top of the tool
    panel and search by data type or tool name to make finding these
    easier, for example "Cut" or "Join" or "Tabular" - these tools have
    the names you would probably expect them to have and tool help is
    directly on each form. Our 101 tutorial also would be a good
    introduction for an overview: https://main.g2.bx.psu.edu/u/aun1/p/galaxy101

Hopefully this gives you some helpful information to work with,

Jen
Galaxy team


On 4/8/13 7:21 PM, Veranja Liyanapathirana wrote:
 
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, 
>Veranja  
>  
>From: Veranja Liyanapathirana mailto:veranj...@yahoo.com
>To: galaxy-user mailto:galaxy-user@lists.bx.psu.edu 
>Sent: Saturday, 6 April 2013, 23:13
>Subject: Error in creating Depth of Coverage files after Bowtie for Illumina 
>alignment 
>  
>
>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 mailto:veranj...@yahoo.com
>To: galaxy-user mailto: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)    
>
>     
>
>   
> 
>
>___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
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-- 
Jennifer Hillman-Jackson
Galaxy Support and Training http://galaxyproject.org/  
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  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:

  http://lists.bx.psu.edu/listinfo/galaxy-dev

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

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To search Galaxy mailing lists use the unified search at:

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