Hi Debora,
Sorry to hear that you are having problems. We can help get you
going again! Please see below:
On 9/26/13 7:20 AM, garzetti wrote:
Dear all,
I have been looking for an answer to my problem in all the Galaxy
Support resources but with no success. I am sorry if this topic
has been already discussed!
So, I am analyzing MiSeq data on the main Galaxy.
I have Fastq files from 4 paired-end samples. After having checked
the quality with FastQC and groomed them, I have performed a BWA
mapping, filtered the results and converted the SAM to BAM files
(for each sample separately). I have then called SNPs with
Freebayes and SAMtools, encountering problems in both cases.
1) SAMtools: if I run the Generate pileup tool, then the Filter
pileup doesn't recognize any valid format in the files I have in
my History and I cannot go on with the analysis. Why is that? What
can I do?
Make sure that the output format is set as "pileup" and the tool
will accept the input. Click on the pencil icon to make the datatype
assignment change.
http://wiki.galaxyproject.org/Support#Tool_doesn.27t_recognize_dataset
Note that Mpileup has an option to produce .bcf format, and that is
not the same as pileup. If you have selected that type of output,
then either re-run the tool with options that create pileup format,
or convert bcf -> vcf and use one of the tools that work with vcf
format to work with your data downstream from there.
2) I have performed variant calling with Freebayes on single BAM
files and on one merged BAM files from all my four BWA mapping
files. In all cases, the last column is "unknown", while it should
be the name of my sample. This is not a big deal for the single
vcf files, but from the merged BAM file, I cannot discriminate
from which sample the SNPs were detected. I think there is a
problem in the BAM files which are not properly indexed. Also
Freebayes needs an RG tag.
Is there a tool in Galaxy I can use to index BAM files, adding the
RG tag?
The tool "
NGS: Picard (beta) -> Add or Replace Groups" can be used to
annotate SAM/BAM files. This tool can be a bit picky about formats,
so just watch for that if you get an error.
Quick tip: You can click on the bug icon on failed
datasets to see the complete error message and it will often tell
you exactly what is wrong so that you can correct it (this doesn't
automatically submit a bug, which is good to know when you are in a
hurry at night or on weekends or just want to troubleshoot
yourself). You can use this on any error dataset to get more
information if the dataset's "i" info button's stderr/stdout links
or attributes "Info" field does not provide enough details. =>
This functions on servers that have bug reporting enabled (the
public Main server does, and this is straightforward to configure on
local/cloud instances, including your own, even if you use one for
small local file manipulations or file backup/storage (very handy
& key file backups are always a good idea, when doing analysis
in general, anywhere). See the Admin wiki section for more.
Going forward, there is a short screencast about the Learning
resources in Galaxy here in a Page. It will be uploaded to Vimeo
sometime in the next 24 hrs, and will be likely updated to include
the very latest as the infrastructure updates on Main settle out in
the next weeks or so, but for now here is the link: Click on the
"Learning Resources" graphic to launch the quick tour:
https://main.g2.bx.psu.edu/u/galaxyproject/p/screencasts-usegalaxyorg
Galaxy team's Vimeo account: http://vimeo.com/channels/581769
We are uploading all of our vids, old & new, right now and over
next few days. We really like and hope our user's do too and follow
along. The public Main server will have direct links to this
content, in the center home page, soon as part of the "New &
Improved" Galaxy experience! I won't give an ETA, as this is in
progress, but can hint that soon == expected very soon. (!)
Good luck and let us know if you need more help,
Jen
Galaxy team
I hope someone can help me!
Thank you very much!
Debora
--
Jennifer Hillman-Jackson
http://galaxyproject.org