Keith Satterley, Bioinformatics Division, WEHI

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Bioinformatics Seminar 13/11/07

• Keith Satterley, Bioinformatics Division, WEHI

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Summary

• GABOS Get A Bit Of Sequence.
• GAFEP Get A Few Exon Primers.
• Functions and Facilities
• WEB interface.
• Command Line Interface.
• Data Management
• Genome data.
• Result data.
• Tools Used
• Perl
• HTML
• PHP
• Javascript
• Availability.
• Future Work.

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• GABOS version 1 is at http//unix28.alpha.wehi.edu
  .au/bioinformatics/gabos
• WEB Page version 1 limitations
• Exons, DNA, Transcripts available.
• Genomes are a hard coded list of latest version
  data only.
• Annotation File is a hard coded list covering all
  genomes.
• Chromosome selection was a list of the common
  chromosome filenames.
• Data Files Availability
• All data has been downloaded from UCSCs download
  site. It is described at
• http//hgdownload.cse.ucsc.edu/downloads.html and
  can be ftp downloaded from
• ftp//hgdownload.cse.ucsc.edu/goldenPath/
• Genome data is stored on the WEHI Disk Server
  accessible from
• WEHI Unix computers
• /home/users/lab0605/Bioinformatics/databases/genom
  es/UCSC
• WEHI Windows computers map a network drive to
• \\unix33\bioinformatics
• WEHI Macintoshes Connect to Server at
• smb//unix33/Bioinformatics

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• Genomes at WEHI
• Jul 24 0105 canFam -gt canFam2
• Jul 23 1516 canFam1
• Jul 23 1516 canFam2
• Jul 22 0117 danRer -gt danRer4
• Jul 23 1033 danRer3
• Jul 23 1520 danRer4
• Nov 6 0110 dm -gt dm3
• Nov 5 1637 dm3
• Jul 22 0117 galGal -gt galGal3
• Jul 20 1727 galGal2
• Jul 23 1011 galGal3
• Jul 22 0117 hg -gt hg18
• Jul 23 1029 hg17
• Jul 23 1029 hg18
• Aug 24 0110 mm -gt mm9
• Jul 23 1030 mm7
• Aug 23 1450 mm8
• Aug 23 1812 mm9

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• Chromosome data Files
• Aug 23 1409 chr9_random.fa
• Aug 23 1409 chrM.fa
• Aug 23 1409 chrUn_random.fa
• Aug 23 1414 chrX.fa
• Aug 23 1414 chrX_random.fa
• Aug 23 1414 chrY.fa
• Aug 23 1416 chrY_random.fa
• Jul 23 1611 chr9.fa
• Jul 23 1611 chrM.fa
• Jul 23 1613 chrNA_random.fa
• Jul 23 1614 chrUn_random.fa
• Jul 23 1614 md5sum.txt
• Jul 23 1614 README.txt
• Jul 23 1616 scaffoldNA_random.fa
• Jul 23 1616 scaffoldUn_random.fa
• Jun 22 0405 chr2L.fa

• Annotation Data Files

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• Data Management
• Amount of data
• How many genomes local? currently 10 96GB.
• 19 Vertebrates available 9 sequence only.
• 15 Insects, 5 Nematodes 4 others available.
• How many versions of each? mm7, mm8, mm9?
• 2 or 3 of each?
• Chromosome data 10-50 per genome.
• Annotation data 5-10 per genome version
• RefSeq, genscan, mgc, xenoRef, uniGene, refFlat,
• ESTs. mRNAs
• Up to date data!
• Tool currently being written to nightly check
  UCSC
• Download, unpack and sort annotation files.

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• GABOS Sequence Retrieval Features
• Specify Search Criteria as either
• Gene Name List
• as in Annotation Files
• NM_001037759,NM_145692, NM_027033, NM_013715 as
  in RefSeq.txt
• Sgk3, 4930418G15Rik, Cops5, Sulf1 as in
  RefFlat.txt
• Chromosome Sequence Range specification.
• Chr1013,500,000 - 14,550,000
• This will select all genes in this region that
  are defined in the annotation file(s) specified.
• Exons (incl. EST exons), Transcripts of Genes or
  straight DNA sequence can be retrieved.
• Specify either strand or both strands.

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• Extra Sequence Parameters
• Range of bases in data object (for e.g. bps in an
  Exon)
• 1-e all, base 1 to the end base (the default)
• 1-10 bases 1 to 10
• 10-e base 10 to end base in object.
• Range of objects requested. (for e.g. a range of
  Exons)
• 1-e all exons (the default)
• 1-3 exons 1 to 3.
• 1 first exon only
• e last exon only
• Possible Extensions
• (e-3)-e last three objects (or bases)

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• GABOS Extras
• Specify the line length of the FASTA output file.
• Output Sequence Lines ONLY.
• Output Fasta Description Lines ONLY.
• Concatenate ALL Sequences.
• Concatenate ONLY Sequence from a DNA object (Each
  genes exons concatenated for example).
• String of characters to be inserted BEFORE each
  DNA object.
• String of characters to be inserted AFTER each
  DNA object.
• Specify flanking bases.
• Show co-ordinates relative to Chromosome, Exon,
  Transcript
• Uses either RefSeq or Browser gene names in
  refFlat.txt
• GAFEP (Get a Few Exon Primers)
• Use output of GABOS to find primers around each
  exon.

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• GABOS Command Line Version (CLI).
• Same code. Program detects environment and
  adjusts accordingly.
• CLI use of GABOS caters for programmatic use of
  the tool as part of other tasks.
• For eg. Collecting 5000 bases before a transcript
  and 5000 into the transcript to be used for
  promoter/regulation searching for thousands of
  genes.

CLI Eg. gabos -afile refFlat.txt -genome mm9
-seqrange 4,482,560-4,483,185 -chr 1 -pre 420
-post 420 fastaonly gtmy_results.fa Options can
be in any order. Output can be redirected to a
file as shown. A file of gene names could be used
as input instead of a chromosome sequence
range. gabos help lists all options.
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• CLI additional abilities.
• Gene lists read from a file or piped in.
• Debugging options available.
• Specification of alternate locations for
• (enables use of program at other sites without
  modification.)
• Annotation files.
• Genome data files.
• Checks if data files are latest version and
  updates if not (To be replaced with upgraded
  procedure).

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GABOS Command Line options
All GAFEP programs can also be run at the command
line. In particular Combine_overlapping_exons, C
reate_primers1, Create_primers2
, Makep3i, P3out2tab.

• -addends,
• -addstarts,
• -dnas,
• -basedirs,
• -genomes
• -afiles,
• -adirs,
• -gdirs,
• -check!
• -names,
• -nameps,
• -namefs,
• -chrs,
• -seqranges,
• -strands,
• -dataobjects,
• -objectranges,

• -baserange
• -seqonly,
• -fastaonly,
• -linelengthi,
• -relatives,
• -prei
• -posti
• -v!
• -debug1i,
• -debug2i,
• -debug3i,
• -debug4i,
• -debug5i,
• -debug6i,
• -debugalli,
• -hhelp?,
• -version

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• Demo of GABOS version 2.
• http//unix28.alpha.wehi.edu.au/bioinformatics/gab
  os/testing_index.php
• Improvements
• Automatically reads genomes available
• Automatically shows chromosome data for genome
  selected.
• Automatically shows Annotation data files for
  genome selected.
• Includes ability to read EST data files.
• Uses alternate gene name in refFlat.txt.
• Faster processing of large data files
  using/making presorted versions.

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• GAFEP Get A Few Exon Primers.
• This is a suite of programs.
• Combines overlapping exons into one CExon.
• Displays Primer3 options and collects choices.
• Creates input files for Primer3 in the required
  format.
• Runs Primer3, displays output on the web page and
  reformats the output suitable for pasting into
  Excel.
• The same code runs from the web interface or
  from a Command Line Interface.

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Combining Exons to reduce number of primers
needed.
1
CExon
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• Demonstration of GAFEP

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GAFEP Output
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• An example application
• Ben Kiles lab are using GABOS/GAFEP to create
  primers to search for variations in sequence
  caused by the ENU mutations in mice.

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Random chemical mutagenesis in the mouse
N-ethyl-N-nitrosourea (ENU)

• Alkylating agent
• Point mutagen
• Efficiently mutates mouse spermatogonial stem
  cells
• Male mice treated with ENU produce offspring
  heterozygous for ENU-induced mutations at the
  rate of 1 mutation per 1.5 megabases

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Phenotyping screen measuring platelet number
Blood test
Mutant offspring
Platelet counts
Platelet count x103/uL
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Mapping strategy for dominant mutations
Affected
Wild-type
C57BL/6
X
1st Outcross
Balb/c
m
X
F1 Generation
2nd Outcross
Affected
Unaffected
F2 Generation
m
m
m
m
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Mapping strategy for dominant mutations

• Genome-wide scan with 80-100 microsatellites
• 20 affected and 20 unaffected animals
• Result mutation assigned to a chromosome
• 2. Fine mapping
• 200-1,000 informative meioses, genotyped with
  SSLPs at increasing density
• Result candidate interval refined to 1-3 Mb
• Issues
• Recombination cold spots
• Polymorphism deserts

SNP density map of mouse chromosome 1 (C57BL/6 v
129Sv)
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Candidate intervals
Heaven
Hell
Chromosome 2 20-21 Mb
Chromosome 11 70-71 Mb
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Candidate gene sequencing

• Prioritize candidates for sequencing on the basis
  of
• Known function
• Homology to other genes of known function
• Tissues expression pattern
• Domain structure
• Exhaustive literature searches..

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Candidate gene sequencing
1. Automated PCR primer design
Robotic liquid handling
2. Genomic PCR
In-well template clean-up
3. Direct amplicon sequencing
4. Capillary electropheresis
5. Sequence analysis
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• Tools used to develop GABOS/GAFEP
• Perl programming language for all programs.
• Web interface
• HTML coding
• PHP inserted into HTML and processed by the
  webserver before the HTML is processed by the
  webserver.
• Javascript processed by the clients web browser
  (Mozilla Firefox or Safari for example)

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WEHI Computing Layout
Unix Server unix28
php processed here
Webserver apache
Client Mac, Windows.
html produced here
Browser Firefox,IE
wan/lan
html processed here
Javascript acts here In response to user
nfs
Unix28 disk GABOS/GAFEP
unix33
Display of GABOS/GAFEP here
Genome DATA
ftp
UCSC
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• Web Interface Debugging tools
• Firefox Error Console
• Firebug Addin to Firefox

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• Future Work
• Short term
• Finalize GABOS version 2
• Transcript, DNA working
• Complete data download maintenance program
• Automate sorting of annotation files and modify
  GABOS to be aware of sorted/non-sorted data and
  act accordingly.
• Include ability to retrieve RNA data
• Will run on any unix server not just unix28.
• Web Interface available on WEHIs public server.
• Source code will be made freely available.
• Longer Term
• Retrieve data for utrs, others?
• Provide web interface access to annotation files.
  
• Remove need for BioPerl to be installed.

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• Aknowledgements
• Bioinformatics Division
• Terry Speed Gordon Smyth for the opportunity to
  pursue this project in an excellent environment.
• All others in Bioinformatics for many and varied
  help.
• WEHI ITS
• Nick Tan, Jakub Szarlat for Unix help.
• Dung Tran, Scott Wood for network help.
• Tri Le and John Nguyen for MS windows support.
• Tony Kyne others in ITS for many questions
  answered.
• Molecular Medicine
• Doug Hilton, Ben Kile for explaining their needs.
• Users for their feedback.
• Kylie Greig, Adrienne Hilton, Greg Hather,
  Carolyn de Graaf