New subject: UCSC->EMBOSS/fuzznuc->UCSC workflow?

18 May 2011

      Hello Curtis,
The BED extraction data can be resolved in Galaxy. Pull out the whole 
gene and then modify the coordinates in Galaxy to be 10k upstream.
To be clear - this coordinate data is going to be used to transform the 
coordinates in your current fuzznuc output that is transcript-based to 
be genome-based. The coordinates are not input for fuzznuc - the are 
used after fuzznuc is run on the fasta file, in order to covert the 
result coordinates only.
This page in the UCSC wiki has a good description of how the UCSC 
coordinates are organized.
http://genomewiki.ucsc.edu/index.php/Coordinate_Transforms
The output format for fuzznuc is documented in the tool's help - the 
last line on the tool form has a link.
Hopefully this helps to clear up the suggested processing,
Thanks,
Jen
Galaxy team
On 5/17/11 2:08 PM, Robert Curtis Hendrickson wrote:
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Jennifer,
I tried getting data from UCSC as .BED - two issues:

Unlike "get sequence", I can no longer specify how far upstream I

want - it's EITHER "whole gene" (what's the definition of that!!!) OR
#bp_upstream OR exons OR introns -- with get seq those are not mutually
exclusive - I happen to want the genomic region (5'UTR, exons, introns
3'UTR + 10kbp upstream of 5'UTR)

fuzznuc does not recognize BED as a valid input format. So, I can't

run fuzznuc because my BED file doesn’t' show up in the pulldown.
Indeed, BED files are just annotation, they don’t carry any sequence.
Have I mis-understood your directions?
Regards,
Curtis
-----Original Message-----
From: Jennifer Jackson [mailto:jen@bx.psu.edu]
Sent: Tuesday, May 17, 2011 11:23 AM
To: Robert Curtis Hendrickson
Cc: 'galaxy-user@lists.bx.psu.edu'
Subject: Re: [galaxy-user] UCSC->EMBOSS/fuzznuc->UCSC workflow?
Hello Curtis,
No need to use the fasta headers from your original fasta file.
To obtain the coordinates in BED format: using "Get Data -> UCSC main"
again to link to the UCSC Table browser, set the same selection criteria
as for the original fasta sequence, only change the output type to be
"BED" (instead of "sequence"). Once in your Galaxy history, this format
will be easier to work with.
Best,
Jen
Galaxy team
On 5/16/11 9:04 PM, Robert Curtis Hendrickson wrote:
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Jennifer,
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Thanks for the outline. I'll try that approach.
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However, it seems rather painful to have to join the fuzznuc output
back to the original fasta to get at the header information that really
should have been passed along. It would see that there must be a way to
get the data out of UCSC without that space in the fasta header, so that
the chromosome& genomic location get correctly preserved in the fuzznuc
output. Failing that, is there an easy text manipulation that would
convert that fasta header space to a "|"?
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Regards,
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Curtis
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-----Original Message-----
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From: Jennifer Jackson [mailto:jen@bx.psu.edu]
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Sent: Monday, May 16, 2011 6:50 PM
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To: Robert Curtis Hendrickson
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Cc: 'galaxy-user@lists.bx.psu.edu'
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Subject: Re: [galaxy-user] UCSC->EMBOSS/fuzznuc->UCSC workflow?
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Hello Curtis,
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The coordinates of your match are with respect to the fasta sequence,
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not with respect to the reference genome. Only data mapped to the
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reference genome can be viewed in the UCSC Browser
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You will need to calculate from the position of the match in the fasta
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sequence back through to the reference genome.
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One suggested way to do this:
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a) Merge together the original genomic coordinates of the 2kb regions
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with each line of output from fuzznuc. Use the original source fasta
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sequence name as the common key for the merge. If both data are in BED
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format, that would be ideal and make the following steps possible. You
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may need to split the file based on whether the original fasta sequence
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came from the positive or negative strand to run "b" and "c" below
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separately.
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b) Use "Text Manipulation -> Compute an expression on every row" to
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create new coordinates. For example, if your data is on the positive
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strand, and base 1 in your fasta file was genomic coordinate 100, and
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the alignment from fuzznuc started at base 5 (local coordinate == "4" if
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in BED format with a zero-based start), then the new genomic start
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coordinate would be [100 + 4)] = 104. Do this for both start and stop.
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c) Adjust the logic for "b" if any of your original fasta sequences are
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from the negative strand, on the negative strand portion of your data
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("b" would be run on just the positive strand portion of your data).
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d) arrange/cut the resulting file down into a standard BED format to
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remove the local coordinates and keep the genomic coordinates, using the
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original chromosome names.
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e) once the logic for the calculations is worked out, save the process
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into a workflow for use again.
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Hopefully this helps,
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Best,
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Jen
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Galaxy team
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On 5/13/11 9:32 AM, Robert Curtis Hendrickson wrote:
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Folks,
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I wanted to scan the 2kb upstream of a list of human gene isoforms
for TFBS using fuzznuc. I was able to
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"Get Data"> "UCSC Main"> "As sequence" and get my sequences
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"EMBOSS"> fuzznuc ran fine, and output the hits
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HOWEVER, fuzznuc lost the genomic position information that UCSC has
put after a space in the sequence headers of the FASTA file. It only
provided offsets within the fasta.
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http://main.g2.bx.psu.edu/u/curtish-uab/h/ucsc-fuzznuc-ucsc-broken
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Thus, when I converted the fuzznuc output back to a BED file and
tried to visualize the hits in UCSC browser, it failed with "invalid BED
File".
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I tried fuzznuc with output: seqtable, feattable and gff3, but in
all cases the genomic position was missing, and being a bit of Galaxy
novice, I couldn't figure out how to get the output back to UCSC to
visualize the hits.
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Can anyone tell me how to link up these tools correctly, or share a
history with some other tool set that accomplishes this goal?
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Regards,
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Curtis
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Research Associate
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Center for Clinical and Translational Science
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University of Alabama at Birmingham
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--
Jennifer Jackson
http://usegalaxy.org
http://galaxyproject.org
-- 
Jennifer Jackson
http://usegalaxy.org
http://galaxyproject.org