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1 Keith Satterley, Bioinformatics Division, WEHI Bioinformatics Seminar 13/11/07.

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1 1 Keith Satterley, Bioinformatics Division, WEHI Bioinformatics Seminar 13/11/07

2 2 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.

3 3 GABOS version 1 is at 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 UCSC’s download site. It is described at: and can be ftp downloaded from: –Genome data is stored on the WEHI Disk Server accessible from: WEHI Unix computers –/home/users/lab0605/Bioinformatics/databases/genomes/UCSC WEHI Windows computers – map a network drive to: –\\unix33\bioinformatics WEHI Macintoshes – Connect to Server at: –smb://unix33/Bioinformatics

4 4 Genomes at WEHI: –Jul 24 01:05 canFam -> canFam2 –Jul 23 15:16 canFam1 –Jul 23 15:16 canFam2 –Jul 22 01:17 danRer -> danRer4 –Jul 23 10:33 danRer3 –Jul 23 15:20 danRer4 –Nov 6 01:10 dm -> dm3 –Nov 5 16:37 dm3 –Jul 22 01:17 galGal -> galGal3 –Jul 20 17:27 galGal2 –Jul 23 10:11 galGal3 –Jul 22 01:17 hg -> hg18 –Jul 23 10:29 hg17 –Jul 23 10:29 hg18 –Aug 24 01:10 mm -> mm9 –Jul 23 10:30 mm7 –Aug 23 14:50 mm8 –Aug 23 18:12 mm9 –Jul 22 01:17 monDom -> monDom4 –Jul 23 10:32 monDom4 –Jul 22 01:17 panTro -> panTro2 –Jul 23 10:32 panTro1 –Jul 23 10:33 panTro2 –Jul 22 01:17 rheMac -> rheMac2 –Jul 25 02:32 rheMac2 –Jul 22 01:17 rn -> rn4 –Jul 23 10:33 rn3 –Jul 23 10:33 rn4 More can be downloaded as requested.

5 5 Chromosome data Files: – Aug 23 14:09 chr9_random.fa – Aug 23 14:09 chrM.fa – Aug 23 14:09 chrUn_random.fa – Aug 23 14:14 chrX.fa – Aug 23 14:14 chrX_random.fa – Aug 23 14:14 chrY.fa – Aug 23 14:16 chrY_random.fa – Jul 23 16:11 chr9.fa – Jul 23 16:11 chrM.fa – Jul 23 16:13 chrNA_random.fa – Jul 23 16:14 chrUn_random.fa – Jul 23 16:14 md5sum.txt – Jul 23 16:14 README.txt – Jul 23 16:16 scaffoldNA_random.fa – Jul 23 16:16 scaffoldUn_random.fa – Jun 22 04:05 chr2L.fa – Jun 22 04:05 chr2LHet.fa – Jun 22 04:05 chr2R.fa – Jun 22 04:05 chr2RHet.fa – Jun 22 04:05 chr3L.fa – Jun 22 04:05 chr3LHet.fa Annotation Data Files:

6 6 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: per genome. Annotation data: 5-10 per genome version –RefSeq, genscan, mgc, xenoRef, uniGene, refFlat, –EST’s. mRNA’s … –Up to date data! Tool currently being written to nightly check UCSC Download, unpack and sort annotation files.

7 7 GABOS Sequence Retrieval Features –Specify Search Criteria as either: Gene Name List –as in Annotation Files »NM_ ,NM_145692, NM_027033, NM_ as in RefSeq.txt »Sgk3, G15Rik, Cops5, Sulf1 as in RefFlat.txt Chromosome Sequence Range specification. –Chr10: 13,500, ,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.

8 8 Extra Sequence Parameters –Range of bases in data object (for e.g. bps in an Exon) 1-e = all, base 1 to the e nd base (the default) 1-10 = bases 1 to e = base 10 to e nd 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)

9 9 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 gene’s 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.

10 10 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 >my_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.

11 11 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 ).

12 12 GABOS Command Line options: -addend:s, -addstart:s, -dna:s, -basedir:s, -genome:s -afile:s, -adir:s, -gdir:s, -check! -name:s, -namep:s, -namef:s, -chr:s, -seqrange:s, -strand:s, -dataobject:s, -objectrange:s, -baserange -seqonly, -fastaonly, -linelength:i, -relative:s, -pre:i -post:i -v! -debug1:i, -debug2:i, -debug3:i, -debug4:i, -debug5:i, -debug6:i, -debugall:i, -h|help|?, -version All GAFEP programs can also be run at the command line. In particular: Combine_overlapping_exons, Create_primers1, Create_primers2, Makep3i, P3out2tab.

13 13 Demo of GABOS version 2. –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.

14 14 GAFEP = Get A Few Exon Primers. This is a suite of programs. 1.Combines overlapping exons into one “CExon”. 2.Displays Primer3 options and collects choices. 3.Creates input files for Primer3 in the required format. 4.Runs Primer3, displays output on the web page and reformats the output suitable for pasting into Excel. 5.The same code runs from the web interface or from a Command Line Interface.

15 15 Combining Exons to reduce number of primers needed CExon Exon

16 16 120bp Short Exons Pad out short exons to 300 bp. Primers in flanks Add a 70 bp. cushion Add 200bp flanks 120

17 17 900bp Long Exons Primers in flanks Add 200bp flanks bp overlap 485bp Split Add a 70 bp. cushion

18 18 Demonstration of GAFEP

19 19 GAFEP Output

20 20

21 21 An example application: Ben Kile’s lab are using GABOS/GAFEP to create primers to search for variations in sequence caused by the ENU mutations in mice.

22 22 Random chemical mutagenesis in the mouse 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 N-ethyl-N-nitrosourea (ENU)

23 23 Phenotyping screen: measuring platelet number Platelet counts Platelet count x10 3 /uL Plt16 and Plt20 cause dominant thrombocytopenia Mutant offspring Blood test

24 24 Mapping strategy for dominant mutations m mmm m X X F1 Generation F2 Generation Affected C57BL/6 Balb/c m Wild-type 2nd Outcross 1st Outcross AffectedUnaffected

25 25 Mapping strategy for dominant mutations 1. Genome-wide scan with 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)

26 26 Candidate intervals Chromosome 2: Mb Chromosome 11: Mb HeavenHell

27 27 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…..

28 28 Robotic liquid handling 2. Genomic PCR 3. Direct amplicon sequencing 4. Capillary electropheresis 1. Automated PCR primer design 5. Sequence analysis In-well template clean-up Candidate gene sequencing

29 29 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)

30 30 Unix Server = unix28 Webserver = apache Client = Mac, Windows. Browser = Firefox,IE … Display of GABOS/GAFEP here Genome DATA unix33 UCSC nfs ftp Unix28 disk GABOS/GAFEP wan/lan Javascript acts here In response to user html produced here php processed here html processed here WEHI Computing Layout

31 31 Web Interface Debugging tools –Firefox Error Console –Firebug Addin to Firefox

32 32 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 WEHI’s 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.

33 33 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 …

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