Woods Hole 2012 Zebrafish Bioinformacs Lab All materials can be downloaded from http://faculty.ithaca.edu/iwoods/docs/wh/! ! (or) hp://goo.gl/1bnOF ! Ian G. Woods August 2012 Task 1: High resolu1on mapping, sequencing, and expression Overview: From a rough map position, refine the critical interval via (virtual) high resolution mapping with additional markers. Query the critical interval in the zebrafish genome for potential candidate genes. Find expression patterns online for these candidates. Design primers to sequence candidate genes for the mutagenic lesion or for additional SNPs to use in mapping. Task 2: Clone candidate enhancer/promoter sequences to create a transgenic reporter line Overview: Iden7fy the translaonal start site of a gene of interest. Obtain ~6kb of sequence upstream of this site. Design PCR primers that will amplify this region, and clone it in-frame with GFP in a tol2 expression vector. Iden7fy BACs for use in creang reporter constructs via homologous recombinaon or gap repair. Iden7fy evolu7onarily conserved sequences from other organisms to uncover poten7al regulatory regions around your gene of interest. Task 3: Morpholinos, rescue, and expression Overview: Find the zebrafish ortholog of your favorite gene. Find its locaon in the genome, locate the ATG, and iden7fy the exon-intron boundaries. Design two 25-mer morpholino sequences that target (1) the ATG and (2) an exon- intron boundary. Iden7fy an orthologous gene in another species for use in rescue experiments to control for morpholino specificity. Align this sequence with your morpholinos to determine degree of poten7al ac7vity. Obtain a full- length clone of the zebrafish gene (via RTPCR or clone collec7ons) for use in overexpression experiments or expression analyses via in situ hybridizaon. Task 4: Iden1fying zebrafish transcripts via Batch sequence retrieval and BLAST Overview: Mine OMIM (Online Mendelian inheritance in Man) for genes related to Sonic Hedgehog. Get amino acid sequences for these genes, and iden7fy (via blast) the zebrafish orthologs for these proteins. Use a simple script to parse the blast results to see where the genes are located in the zebrafish genome. Finally, find out where a few of these genes are expressed (via zfin). Requirements: UNIX terminal, perl (both nave on MacOSX) Detailed protocols and results Task 1: High resolu1on mapping, sequencing, and expression Overview: From a rough map position, find more SSLP markers to test for polymorphisms in your mapping cross. Query the critical interval in the zebrafish genome for potential candidate genes. Find expression patterns online for these candidates. Design primers to sequence candidate genes for the mutagenic lesion or for additional SNPs to use in mapping. ZFIN home http://www.zfin.org! Click on “Genetic Maps” ZFIN Gene7c Maps Browser Uncheck all but “MGH” and enter marker symbol ZFIN MGH map viewer Zoom out as far as possible ZFIN Map Viewer Z3057 is at ~38 cM. Z13936 is at ~85 cM. Plenty of markers to follow up on. Find primer sequences for Z15270 Find addi7onal SSLPs to test http://www.ensembl.org! Find addi7onal SSLPs to test enter Z15270 into the search box and hit go Follow the links . Ensembl Marker Report Viewer Ensembl Chromosome View Zoom out a bit Configuring Tracks in Ensembl Zoom out in region what kind of gene is LOC563432? Ensembl gene view Click the ‘Orthologues’ link Ensembl gene view Scroll down a bit . looks like a phosphodiesterase Record for rras2 links to expression pattern – click on External References Link out to ZFIN ZFIN expression data Click on ‘Directly submi?ed expression data’ link ZFIN expression data rras2 expression – consistent with a role in muscle development? ZFIN home http://www.zfin.org! Follow link for Genes / Markers / Clones ZFIN gene search Type in rras2 ZFIN gene search results Click on “1 Gene” ZFIN gene record Scroll down . ZFIN gene record Follow link for RNA sequence GenBank gene record Scroll down . GenBank gene record Copy Sequence to Clipboard – note cds starts at 240 UCSC Genome Home Click on the “BLAT” tab UCSC BLAT search Paste in your sequence, and select “Zebrafish” from the Genome menu UCSC BLAT results Follow “details” link for the top hit UCSC BLAT results Light blue = exon boundaries Select about 600-800b of genomic sequence around the first exon Primer3 Home http://frodo.wi.mit.edu/primer3/! Choose size range of 500-600 Primer3 Results BLAST 2 Sequences http://blast.ncbi.nlm.nih.gov/Blast.cgi... ‘nucleotide blast’! Paste in wildtype and mutant sequences BLAST 2 Result Scroll down . BLAST 2 Result There are two SNPs . dCAPS Home http://helix.wustl.edu/dcaps/dcaps.html! Paste about 40b of wildtype and mutant sequence in flanking each SNP dCAPS results – SNP1 Not much luck, but can introduce differen7al restric7on sites via primers dCAPS results – SNP2 Plenty of enzymes from which to choose Do the SNPs affect coding? BLAST mutant gDNA vs. cDNA Do the SNPs affect coding? Query = mutant sequence; Subject = GenBank refseq SNPs are not in coding sequence Task 2: Clone candidate enhancer/promoter sequences to create a transgenic reporter line Overview: Iden7fy translaonal start site of gene of interest. Obtain 6kb of sequence upstream of this site. Design PCR primers that will amplify this region, and clone it in-frame with GFP in a tol2 expression vector. Iden7fy BACs for use in creang reporter constructs via homologous recombinaon. Iden7fy evolu7onarily conserved sequences from other organisms to uncover poten7al regulatory regions around your gene of interest. ZFIN home http://www.zfin.org! Follow link for Genes / Markers / Clones ZFIN Gene Search type in ‘scube2’ ZFIN Search Result Follow link for Gene ZFIN Gene record Scroll down a bit . ZFIN Gene record Zfin localizes this gene to Chr. 7 GenBank Gene record Scroll down . GenBank Gene record Find the heading for “CDS” = coding sequence GenBank Gene record The “atg” (translaonal start) is at #106 in this mRNA sequence Ensembl Zebrafish http://www.ensembl.org/Danio_rerio! Enter scube2 into the search box Ensembl text search result Click on “Location” Ensembl Browser – scube2 The transcript is going to the ‘left’ Gene7c vs. physical distance Z15270 ~ 28,880,000 scube2 ~ 29,900,000 Physical distance ~ 1,000,000 Genetic distance = 0.1 cM Total genome = 3000 cM = 1.7 x 109 bp, so about 560,000 bp / cM Genetic distance predicts ~ 56,000 bp away Differences could arise from recombination hotspots/coldspots, and/or errors in sequence assembly scube2 exon 1 scube2 exon 1 – 5000b uh oh .... overlaps another gene scube2 intergenic region Hit ‘export data’ on left part of page scube2 intergenic region – export scube2 intergenic region – export choose ‘text’ scube2 intergenic region – export Primer3 input http://frodo.wi.mit.edu/primer3/! Primer3 output Add enzyme (or gateway) sequences, and clone into your GFP vector Ensembl Zebrafish Home http://www.ensembl.org/Danio_rerio! Enter “scube2” and click “Go”. Ensembl Chromosome view scube2 is split between two sequenced BACS: CU467654 and CU464087 Ensembl Chromosome view Turn on BAC ends track Ensembl Chromosome view No BAC has en7re gene plus putave regulatory sequences, but one has 5’ regions Find a BAC by BLASTing NCBI http://blast.ncbi.nlm.nih.gov/! Click “nucleo7de blast” BLAST scube2 vs. zebrafish nr Enter accession number, click “nr”, and type in “Danio rerio” NCBI BLAST results There are several BACS, but none contain the en7re sequence BLAST2 sequences http://blast.ncbi.nlm.nih.gov/ => ‘nucleotide blast’, ‘Align two sequences’! Place accession numbers for coding sequence on top, and BAC sequence on bo?om BLAST2 result The BAC contains upstream sequence plus about 1500b of coding sequence Zebrafish BAC assembly http://www.sanger.ac.uk/cgi-bin/humpub/chromoview! Zebrafish BAC assembly Aligning genomic sequences with VISTA See exons and conserved noncoding sequences (regulatory?) Iden7fy orthologous sequences Grab pep7de sequence of Scube2 from GenBank record BLAT search at UCSC with pep7de Pull down Tetraodon from Genome menu Which sequence is the true ortholog? Whole genome duplicaon in teleosts makes orthology assignment a bit tricky Which sequence is the true ortholog? Zebrafish Tetraodon S7ckleback Medaka Zebrafish Chromosome #7 = Tetraodon #5 Stickleback #II or #VII Medaka #3 or #18 Clue from conserved synteny scube2 in Tetraodon ZF #7 = Tet #5 scube2 in Tetraodon Zoom out and grab sequence via DNA tab scube2 in S7ckleback Zoom out and grab sequence via DNA tab scube2 in Medaka Zoom out and grab sequence via DNA tab mVISTA Submission Select 4 sequences – upload them on the following page mVISTA upload mVISTA Viewer Probably exons: can adjust parameters to be more or less stringent mVISTA Viewer Conserved non-coding regions => regulatory elements? mVISTA Alignment Zebrafish vs. Tetraodon Task 3: Morpholinos, rescue, and expression Overview: Find the zebrafish ortholog of your favorite gene. Find its locaon in the genome, locate the ATG, and iden7fy the exon-intron boundaries. Design two 25-mer morpholino sequences that target (1) the ATG and (2) an exon- intron boundary. Iden7fy an orthologous gene in another species for use in rescue experiments to control for morpholino specificity. Align this sequence with your morpholinos to determine degree of poten7al ac7vity. Obtain a full- length clone of the zebrafish gene (via RTPCR or clone collec7ons) for use in overexpression experiments or expression analyses via in situ hybridizaon. Entrez Gene Search http://www.ncbi.nlm.nih.gov! Select Gene from the Search menu Entrez Gene Search Search for the first mouse entry Entrez Gene Entry Scroll down . Entrez Gene Entry Click on pep7de (NP_XXX) link Boc GenPept record Copy pep7de sequence to clipboard BLAST home page http://blast.ncbi.nlm.nih.gov/Blast.cgi! Select tblastn Tblastn vs. nr Paste in sequence, select nr, and type Danio rerio into the Organism box Tblastn vs. ests Paste in sequence, select est_others, and type Danio rerio into the Organism box BLAST – access recent searches Click on Request_ID’s to see results Boc vs.