WSSP-10 Chapter 7 BLASTN: DNA vs DNA searches

Transcription

1 WSSP-10 Chapter 7 BLASTN: DNA vs DNA searches 4-3

2 DSAP: BLASTn Page p. 7-1 NCBI BLAST Home Page p. 7-1

3 NCBI BLASTN search page p. 7-2 Copy sequence from DSAP or wave form program p. 7-2

4 Choose a database (nr/nt or est) p. 7-3 Search options (Use defaults) p. 7-4

5 BLASTN progress report (search may take a few minutes) p. 7-5 Format options (use defaults) p. 7-5

6 EX1.10 BLASTN nr/nt database p. 7-6 Graphic report of EX2.09 p. 7-7

7 BLASTN list of matches for EX1.10 p. 7-7 EX2.09 BLASTN p. 7-9

8 Best match to EX1.10 Length of sequence Our Seq. Database Seq. Mismatch Match >gi ref NM_ Zea mays dynein light chain LC6, flagellar outer arm (LOC ), mrna Length=606 Score = 221 bits (244), Expect = 5e-54 Identities = 218/282 (77%), Gaps = 0/282 (0%) Strand=Plus/Plus Query 11 ATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGCAGGCGGAG 70 Sbjct 104 ATGTTGGAAGGAAAGGCGGTGGTGGAGGACACCGACATGCCGGCGAAGATGCAAGCCCAG 163 Query 71 GCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCAAGAGCCTC 130 Sbjct 164 GCGATGTCGGCGGCGTCCAGGGCCCTGGATCGCTTCGACGTCCTCGACTGCCGGAGCATC 223 Query 131 GCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGTGCGTCGTC 190 Sbjct 224 GCGTCCCACATCAAGAAGGAGTTTGACGCGATCCATGGCCCCGGATGGCAATGCGTGGTT 283 Query 191 GGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACTTCCGCCTG 250 Sbjct 284 GGCTCCGGCTTCGGCTGCTACATCACGCACAGCAAGGGGAGCTTCATCTACTTCCGCCTG 343 Query 251 GAGACGCTCCACTTCCTCATCTTCAAAGGCGCGGCCGCTTGA 292 Sbjct 344 GAGTCGCTCAGGTTCCTCGTCTTCAAAGGGGCGGCAGCATGA 385 p. 7-9 Perfect, but short, matches are not usually meaningful >gi emb AL CNS07EFY Human chromosome 14 DNA sequence BAC R-736L22 of library RPCI-11 from chromosome 14 of Homo sapiens (Human), complete sequence Score = 40.1 bits (20), Expect = 4.6 Identities = 20/20 (100%) Query: 189 ttttctgaatattcataata 208 Sbjct: ttttctgaatattcataata

9 Examine the best alignments: Are they significant? 7-9 Mismatches Bad sequence on our part Bad sequence on their part Differences in the sequence of the two organisms Query Sbjct C R E L L I L D A TGT CGT GAA CTC CTA ATT CTC GAC GCC TGT CGT GAA CTT CTG ATC CTT GAT GCA C R E L L I L D A Query 69 ATGAACAAGGAGAAGATTCTGAAGCTGGCGAAGGGCTTCCGGGGGAGGGCGAAGAACTGC 128 Sbjct 242 ATGAACAAGGGAAAGATTTTTAAGCTAGCTAAGGGATTCAGAGGAAGGGCGAAAAATTGC 301 Query 129 ATCCGGATCGCGAGGGAGCGGGTGGAGAAGGCGCTCCAGTACTCGTACCGCGATCGCCGC 188 Sbjct 302 ATAAGGATAGCAAGGGAGAGGGTGGAAAAGGCACTGCAATATTCATACAGGGATCGACGC 361 p. 7-12

10 Small Gaps- alter the reading frame of the protein Query Sbjct C R R T P D P * TGTCGT-CGAACTCCTGATCCTTGA TGTCGTCCGAACTCCTGATCCTTGA C R E L L I L D p An example of a match with and without gaps. Query: 179 TTCGAGCTACCAGATGATC-GATTGGAACAT-T-C--TGTCATTG-AC-CTTC-AGGTAA 230 Sbjct: 4684 TTCGAGCG-CC-GTTAATATGATTACAATATCTACAATATTATTATATGCTTCCAGGTGA 4741 Query: 231 TCAACCATGACCGTGTCAACCGAAACGACGTTATCGGCCGTGCACTATTGAACATGGAGG 290 Sbjct: 4742 TCAATCATGACCGTGTTAACCGTAATGATGTAATTGGCCGTGCCCTTCTTAATATGGAAG 4801 p. 7-13

11 Alignment of the second best match to EX1.10 >gi dbj AK Triticum aestivum cdna, clone: SET5_E05, cultivar: Chinese Spring Length=650 Score = 219 bits (242), Expect = 2e-53 Identities = 211/271 (77%), Gaps = 0/271 (0%) Query 10 GATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGCAGGCGGA 69 Sbjct 78 GATGCTGGAAGGGAAGGCGACGGTGGAGGACACCGACATGCCGGCCAAGATGCAGCTGCA 137 Query 70 GGCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCAAGAGCCT 129 Sbjct 138 GGCCACCTCGGCGGCGTCCAGGGCGCTCGAACGCTTCGACGTCCTCGACTGCCGGAGCAT 197 Query 130 CGCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGTGCGTCGT 189 Sbjct 198 CGCGGCGCACATCAAGAAGGAGTTCGACACGATCCACGGCCCGGGGTGGCAGTGCGTGGT 257 Query 190 CGGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACTTCCGCCT 249 Sbjct 258 GGGCTGCAGCTTCGGCTGCTACTTCACGCACAGCAAGGGGAGCTTCATATACTTCAAGCT 317 Query 250 GGAGACGCTCCACTTCCTCATCTTCAAAGGC 280 Sbjct 318 CGAGTCGCTCCGGTTCCTCGTCTTCAAAGGC 348 p Alignments near the end of the EX1.10 >gi ref NG_ Homo sapiens glypican 4 (GPC4), RefSeqGene on chromosome X Length= Score = 71.6 bits (78), Expect = 6e-09 Identities = 42/44 (95%), Gaps = 0/44 (0%) Query 665 CTAGCTTTTCTTAACaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 708 Sbjct CTTGCTTTTCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA p. 7-14

12 Fill in the table listing the best matches from three different organisms. List Wolffia if there is a match p Use the clone report to obtain more information about the gene p. 7-15

13 3) Perform a BLASTn of the est database Change the database p BLASTn report of the EX1.10 search of the est database p. 7-17

14 Alignment of the best match to EX1.09 from the est search >gi gb GD CCHY28888.g1 CCHY Panicum virgatum callus (N) Panicum virgatum cdna clone CCHY ', mrna sequence. Length=624 Score = 246 bits (272), Expect = 1e-61 Identities = 226/286 (79%), Gaps = 0/286 (0%) Strand=Plus/Minus Query 3 GAGAGAAGATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGC 62 Sbjct 527 GAGACACCATGCTGGAAGGGAAGGCGATGGTGGAGGACACGGACATGCCGGCGAAGATGC 468 Query 63 AGGCGGAGGCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCA 122 Sbjct 467 AGGCGCAGGCGATGGCGGCGGCGTCCAGGGCCCTCGACCGCTTCGACGTCCTCGACTGCC 408 Query 123 AGAGCCTCGCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGT 182 Sbjct 407 GGAGCATCGCGGCGCACATCAAGAAGGAGTTTGACACGATCCACGGCCCCGGGTGGCAAT 348 Query 183 GCGTCGTCGGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACT 242 Sbjct 347 GCGTGGTGGGCTCCAGCTTCGGCTGCTACTTCACGCACAGCAAGGGGAGCTTCATCTACT 288 Query 243 TCCGCCTGGAGACGCTCCACTTCCTCATCTTCAAAGGCGCGGCCGC 288 Sbjct 287 TCCGGCTCGAGTCGCTCAGGTTCCTCATCTTCAAAGGGGCGGCAGC 242 p Fill out the DSAP table of the BLASTn search of the est database p. 7-18

15 Open Question: Why are there differences in the sequences? Query 61 CAAGGTCTAAGTACTGAAAAGGAAAGTCTACTAATTACAAAGAAGTTATTGTTTGTACCT 120 Sbjct CAAGGTCTAAGTACTGAAAAGGAAAGTCCACTAATTACAAAGAAGTTATTGTTTGTACCT Query 121 TTTGTATCAGGGTTTATTAAATTTCAATCTTTATTGCTGAATCCCGAAACAAGGTGATCT 180 Sbjct TTTGTATCAGGGTTTATTAAATTTTAATCTTCATTGCTGAATCCCGAAACAAGGTGATCT 13047