Predicting evolutionarily conserved regions (ECRs) in the Xenopus tropicalis genome using a MultiPipMaker-based bioinformatic strategy OVERVIEW- Method updated and written by Sarah Louie (6/19/08) Initially conceptualized by Hajime Ogino (6/23/07) Laboratory of Robert Grainger Department of Biology University of Virginia 1. Extract genomic sequences of interest from genome assembly data. 2. Build accompanying text files. 3. Generate a multiple genomic sequence alignment. 4. Perform a local realignment of ECR sequences. 5. Identify conserved putative transcription factor binding sites.
ECR browser: identify ECRs using premade alignments Pax6 coding region ECRs Slide 1
ECR browser misses some ECRs; the Pax6CE1 as an example Pax6CE1 Slide 2
Comparison of MultiPipMaker results versusecr Browser and Vista Browser in the detection of the Pax6CE1 enhancer Slide 3
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Identify short evolutionarily conserved regions (ECRs) and perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using rvista. Slide 4
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 5
1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. >Go to Slide 6
>Go to the genome and position of interest Slide 7
>Get orthologous sequences from multiple genomes Get sequence from 1st genome Get orthologous regions in other genomes Slide 8
>Get the genomic DNA sequence from the first genome Slide 9
>Save the genomic DNA sequence as simple text file >xentro2_dna range=scaffold_399:648001-858964 5'pad=0 3'pad=0 strand=+ repeatmasking=none ATATATATATATATATATATATATATATATATATATATATATATACATGA GAATAGATTTCTATGCGAAGTAATATAATAGATTTCTATACTAAGAATAT ATATATATATATATATATATATGTACATTACATGCAATTCATAACAATCA TCACAACAATAACCTTAAATCCCATTACAGTTCAATTACCCAGAACAATA TTTCCCAAGGTAACTCATCTTTTTATGTGTTATTTTTTCCCCCATTACAT TTTAGTCGAATATATGTATATATTTTGTATTTCTATTTTTTTTGCAAATT GATACAAAGCATATGATAAACAATCACCATAGTTACCAGGGTCCTTAGTG CTAATGAGGTCCCCAGTTGCAGCTCAGTAAATTGAAACTATTTACTGCAT AGAAATGTTTTTTTTTCCCGTTTTCTATTTTTCTCCAAAGCTTGTTTACA CCCCTTATTTTTTTTTTTGTGTGTTATTTGGGCTGTGCTGTACACTGACA CTAGTGCTGGCAGGAGGGGGCTGCTGTAGTTCAAGCTACTGAATGTAGAT AATAGTTTCTCCCCCCCCTCTCTCTGTATTCACTTTAAACCATGAACACA AATAGTCGCTTCCACTTGGACTTTTTTTTAATTTATTTTATATTACAGTT TTAAACTTAACCAAGGGCCACAATAAAGGGCCAGGGAACATGTATAATTC ACGGTTTGTTATGCTATTACTTTTCATTCTTCACAAAAAAAAAAAATAGC AAAAACCCCCTTTCTTGTTATAAATTTCTGGAGGTAATTTTGTTACTGGT GTGAGTTGTTAGGGGTTGTAAAACTACACAACAGCTCGGCAGGGGGCTCA ATTGGAAAGAGACAATTACAATAGTGAAAGCATTGAAGAGAGTTGGTTAA AAAGAAAGAAGGGGTAAAATAATGTAATGGCCTGAAATTTCTCTCCAAAA TGCCCTTTCTGTCTCTTTTATGGCAGCAATGAATATCAAGGCTCTATCGA Slide 10
>Convert the genomic DNA sequence to orthologous locations in other genomes of interest (human, mouse, chicken, zebrafish, etc) Slide 11
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 12
>Retrieve the exon and underlay files needed for Multipipmaker from PipHelper Slide 13
>Download the exon and underlay files Slide 14
>Save the exon file as a simple text file Example file for Pax6CE1_Xt containing just the 5 end of coding region : >3748 3977 CE1 > 8183 10964 pax6 8183 8429 exon1 Example file for Pax6CE1_Xt containing all of the Pax6 coding region : < 50114 72318 pax6 50114 50809 UTR 50810 50898 exon 53857 53972 exon 54073 54223 exon 54813 54895 exon 55235 55399 exon 64695 64860 exon 65194 65409 exon 66088 66218 exon 72072 72318 UTR Slide 15
>Save the Pax6CE1_Xt underlay file as a simple text file LightCyan Pax6CE1 3748 3977 LightCyan LightBlue Pax6cds 8183 8429 LightBlue LightYellow Pax6intron 8430 10964 LightYellow Slide 16
>Get the repeat file from RepeatMasker at the ISB Slide 17
>Go to Slide 18
>Download the repeat text file Slide 19
>Save the repeat masker file in simple text Slide 20
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a multiple genomic sequence alignment with high sensitivity using MultiPipMaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 21
>Go to Slide 22
>Input the number of sequences to be aligned 5 Slide 23
>Input the sequence, exon, underlay and repeat files for the base sequence, and just the sequence files from the other genomes Slide 24
>Go to the resulting pip.pdf file for the plot: Pax6CE1 Slide 25
>Go to the resulting alignment in the acgt.pdf file: Slide 26
Example of pip plot for 80 kb surrounding the Pax6 locus with base genome and annotation from the mouse genome: Slide 27
-Ogino, unpubl. Slide 28
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 29
>Perform a local realignment of ECR sequences with ClustalW Use VectorNTI or other basic sequence analysis program or SDSC website for the following tasks: 1. Create sequence files 2. Align (with ClustalW at SDSC website) 3. Shade (with BOXSHADE at SDSCwebsite) Slide 30
>Go to Slide 31
>Add genes to your SDSC Biology Workbench account Slide 32
>Perform local alignment with ClustalW, then perform BOXSHADE on the resulting ClustalW alignment Slide 33
>Save the BOXSHADE results for phylogenetic footprinting Of Transcription Factor Binding Motif (TFBM) analysis Slide 34
Phylogenetic analysis using MultiPipMaker as a more sensitive genome alignment tool for the identification of ECRs OVERVIEW 1) Extract genomic sequences of interest from genome assembly data using the UCSC genome browser. 2) Build accompanying exon, repeat and underlay files using PipHelper and Repeatmasker. 3) Generate a global genomic sequence alignment with high sensitivity using Multipipmaker tools. 4) Perform a local realignment of ECR sequences with ClustalW. 5) Scan for putative transcription factor binding sites using TRANSFAC or user-defined motifs. Slide 35
Scanning for putative transcription factor binding sites- A. Employ the TRANSFAC database (http://www.gene-regulation.com/) OR B. Employ user-defined motifs (http://rsat.ulb.ac.be/rsat/dna-pattern_form.cgi) Slide 36
>Predict TFBMs with the TRANSFAC database at the Gene-regulation website Slide 37
>Transfac search results from P-Match Slide 38
>Check Position weight matrix and references for TRANSFAC database Slide 39
>Enter your own TFBM list and sequences using DNApattern at the RSA tools website Slide 40
>Results of user-defined search using DNA-pattern PatID Strand Pattern SeqID Start End matching_seq Score SEQ_START DR - Pax6CE1_Xt_227bp 1 1-0.00 SEQ_END DR - Pax6CE1_Xt_227bp 227 227-0.00 OTX/PITX D Taakcy Pax6CE1_Xt_227bp 189 194 gtactaatcccaac 1.00 MSX2 D Waatkr Pax6CE1_Xt_227bp 26 31 attaaaatgacgtc 1.00 MSX2 D Waatkr Pax6CE1_Xt_227bp 205 210 ttgctaattgagac 1.00 MSX2 R Waatkr Pax6CE1_Xt_227bp 20 25 attttaatggagag 1.00 TCF/LEF R ctttgww Pax6CE1_Xt_227bp 114 120 tgggctttgtacacc 1.00 RX D ctaattg Pax6CE1_Xt_227bp 204 210 gttgctaattgagac 1.00 CRE DR gacgtc Pax6CE1_Xt_227bp 30 35 aaatgacgtcagct 1.00 SEQ_START DR - pax6ce1_hs_241rev 1 1-0.00 SEQ_END DR - pax6ce1_hs_241rev 241 241-0.00 OTX/PITX D Taakcy pax6ce1_hs_241rev 206 211 gtgctaatccactc 1.00 OTX/PITX R Taakcy pax6ce1_hs_241rev 163 168 gccgtaatcttgtt 1.00 OTX/PITX R Taakcy pax6ce1_hs_241rev 191 196 ttcttaagctttgc 1.00 MSX2 D Waatkr pax6ce1_hs_241rev 222 227 tcgctaattgagag 1.00 SOX D Wwttgww pax6ce1_hs_241rev 67 73 gattttttgttaaaa 1.00 PROSPERO D caynnct pax6ce1_hs_241rev 39 45 cattcactcctcgct 1.00 PROSPERO R caynnct pax6ce1_hs_241rev 106 112 ctcccacccctccaa 1.00 ETS R mggaw pax6ce1_hs_241rev 180 184 gcctcggaaagaa 1.00 TCF/LEF R ctttgww pax6ce1_hs_241rev 127 133 ggctctttgaatcag 1.00 SU(H) D rtgrgar pax6ce1_hs_241rev 110 116 aggggtgggagaagg 1.00 SIP1 R cacct pax6ce1_hs_241rev 200 204 ttagcaccttctt 1.00 RX D ctaattg pax6ce1_hs_241rev 221 227 gtcgctaattgagag 1.00 CRE DR gacgtc pax6ce1_hs_241rev 24 29 caatgacgtcagtg 1.00 SEQ_START DR - Pax6CE1_Mm_240bp 1 1-0.00 SEQ_END DR - Pax6CE1_Mm_240bp 240 240-0.00 OTX/PITX D Taakcy Pax6CE1_Mm_240bp 205 210 gtgctaatccactg 1.00 OTX/PITX R Taakcy Pax6CE1_Mm_240bp 162 167 gccataatcttgtt 1.00 OTX/PITX R Taakcy Pax6CE1_Mm_240bp 190 195 ctcttaagctttgc 1.00 MSX2 D Waatkr Pax6CE1_Mm_240bp 221 226 ttgctaattgagag 1.00 SOX D Wwttgww Pax6CE1_Mm_240bp 68 74 atttttttgttaaaa 1.00 SU(H) D rtgrgar Pax6CE1_Mm_240bp 109 115 gtgggtgggagaagg 1.00 SIP1 R cacct Pax6CE1_Mm_240bp 89 93 atctcacctcaga 1.00 RX D ctaattg Pax6CE1_Mm_240bp 220 226 gttgctaattgagag 1.00 CRE DR gacgtc Pax6CE1_Mm_240bp 24 29 caatgacgtcagcg Slide 41
>Use the Feature Map for visual comparison of predicted TFBMs in each sequence Slide 42
Overlay TFBM search results on ECR alignment to identify predicted binding sites that are also conserved in Xenopus Pax6CE1 Hs Pax6CE1 Mm Pax6CE1 Xt CRE (1) CGGAGCCCCCTTCCCCACCC------AATGACGTCAGTGGCATTCACTCC (1) CGAAGCCCCCATCCCCACCC------AATGACGTCAGCGGCATTCATGCC (1) CGCAGCCCCCCCCCCCTCTCCATTAAAATGACGTCAGCTGTATACA-TCA Pax6CE1 Hs (45) TCGCTCCTTGCCTTTTCTGATTTTTTG-TTAAAACTTTTCTCCGCAGCGA Pax6CE1 Mm (45) AGGCTCCTAGCTTCTTCTGATTTTTTTGTTAAAACTTTTCTCTGAGGTGA Pax6CE1 Xt (50) T--CTCCCTCCCCCTTATGATTTTTTT-TTT----TTTTCTCTAAAACTT Pax6CE1 Hs (94) GATGGGGGTTGGAGGGGTGGGAGAAGGAGCTGATTCAAAGAGC-CAGTGC Pax6CE1 Mm (95) GATGGGGGTTGT--GGGTGGGAGAAGGGGCTGATTGAAAGAGC-CAGTGC Pax6CE1 Xt (93) TTTTT--TTTTGTCGGGAGGGTGTA----------CAAAGCCCACTCTGC Pax6CE1 Hs (143) CCTGGGGGGAAATAGCAACAAGATTACGGCTGCTTCTTTCCGAGGCAAAG Pax6CE1 Mm (142) CCTGGGGGGAAATAGCAACAAGATTATGGCTGCCTCTTTCTTAGGCAAAG Pax6CE1 Xt (131) CCTAGGGAGAAATAGCAACATGATTAGAGCTTCT-----CGGAGCAAAGG OTX/PITX Rx/MSX Pax6CE1 Hs (193) CTTAAGAAGGTGCTAATCCACTCGGTCGCTAATTGAGA---GGTTGTAAA Pax6CE1 Mm (192) CTTAAGAGGGTGCTAATCCACTGGGTTGCTAATTGAGA---GGTTGTAAA Pax6CE1 Xt (176) CATTATTTCGTACTAATCCCAACAGTTGCTAATTGAGACTTGCTTGGAAA Pax6CE1 Hs (240) AA Pax6CE1 Mm (239) AA Pax6CE1 Xt (226) AA Slide 43
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