Due Mar 6 2014 MCB 432 Name key Assignment 6 1 pt each 35 pt total Analyzing Sequences Using NCBI BLAST http www ncbi nlm nih gov blast Blast cgi The data file for this assignment dna1 txt is available in from the course WWW server It is a plain text file with a nucleotide sequence in FASTA format It is a DNA sequence that encodes a protein Go to the NCBI BLAST Home page http www ncbi nlm nih gov blast Blast cgi Follow the link from the BLAST Home page to nucleotide blast Use your computer s Copy and Paste functions to paste a copy of the dna1 definition and sequence into the Enter Query Sequence box 1a In the section of the form entitled Choose Search Set above Program Selection there are 3 radio buttons to select the database to be searched What is the default selection Others nr etc 1b Select Others What is the default other database see the pop up menu Nucleotide collection nr nt Use the menu to set the Database to NCBI Genomes chromosome In the Program Selection section of the form there are radio buttons to select a program version megablast discontiguous megablast or blastn The button selected changes the default settings of some blast parameters Choose megablast Check the box Show results in new window so you can reuse this query page If the Algorithm parameters section of the form is not expanded click the label to make these parts of the form visible Set the following parameters Max target sequences 1000 Short queries Automatically adjust checked Expect threshold 10 Filters and Masks use the defaults Word size should be 28 and Match Mismatch Scores should be 1 2 they are set when you select megablast Click BLAST 2a What is the Request ID RID for your search Varies 2b What is the number of letters nucleotides in this database 1 225 598 357 307 varies 2c What is the number of sequences in this database 50 609 varies 2d From 2b and 2c what is the average length of the sequences in this database 225598357307 50609 4 457 672 varies much less 3 2e How many database sequences match with an E value 10 1 2f How many database sequences match with an E value 1 1 2g What is the bit score Max score for the best match highest total score in the database 699 2h What is the Accession number of the best matching sequence NC 009440 1 2i What is the description of the best matching sequence Metallosphaera sedula DSM 5348 chromosome complete genome 2j This description is not very helpful in understanding the function of the query sequence To see the predicted functions in this part of the genome what is the text following Features hypothetical protein This is not much help Let s see if we can get more matches On the search query page the one that you entered the sequence on change the Program Selection to blastn 3a What is the new default value of Word size 11 3b What are the new default values of Match Mismatch Scores 2 3 Click BLAST The search results will have the same best matching sequence but other data will change 1 If necessary click Search Summary to expand the section Page 2 Assignment 6 Name How many database sequences match with an E value 10 3 2 How many database sequences match with an E value 1 14 this might change slightly What is the Accession number of the second best matching sequence NC 015435 1 What is the E value of the second best matching sequence 4e 24 or 4 10 24 but not 4e 24 What is the description of the second best matching sequence Metallosphaera cuprina Ar 4 chromosome complete genome 4f What do you find for Features for the second best matching sequence None are given Hm Let s see if we can get more matches On the search query page change the Match Mismatch Scores to 1 1 Click BLAST 5a How many database sequences match with an E value 10 3 2 5b How many database sequences match with an E value 1 17 this might change slightly 5c What is the E value of the second best matching sequence 2e 47 or 2 10 47 but not 2e 47 Right direction but not much headway The degeneracy in the genetic code allows the DNA to change much more than the proteins that it encodes To get more matches we need to look at the proteins encoded in the DNA This is a job for BLASTX At the top of the search query page click the blastx tab Set the Database to Reference proteins refseq protein In Algorithm parameters set Max target sequences 1000 leave the rest at defaults Expect threshold 10 Word size 3 Matrix BLOSUM62 Click BLAST 6a How many database sequences match with an E value 10 3 25 6b How many database sequences match with an E value 1 31 6c What is the Accession number of the best matching sequence YP 001192332 1 6d What is the E value of the best matching sequence 1e 84 or 1 10 84 but not 1e 84 6e What is the E value of the second best matching sequence 1e 57 or 1 10 574 but not 1e 57 Scroll down the Alignments 7a What are the descriptions of the 3 best matching sequences without the organism names 1 hypothetical protein Msed 2270 2 pt for all 1 pt for any 2 hypothetical protein Mcup 2016 3 hypothetical protein 7b All of the sequences from Acidianus hospitalis W1 through Sulfolobus acidocaldarius Ron12 I have titles that provide very similar and useful descriptions of the protein function What is the description of the function for Sulfolobus acidocaldarius DSM 639 DNA directed RNA polymerase subunit G 7c Follow the link to the Sulfolobus acidocaldarius DSM 639 sequence What are the source database and accession number NCBI Reference Sequence or just RefSeq YP 255338 or YP 255338 1 7d What is the EC number of the protein 2 7 7 6 7e Back on the search results page all the sequences with 1 E value 10 26 have effectively the same 2 word description What is that description hypothetical protein 8 Given analyses in this assignment in 10 words or less what is the function of his protein DNA directed RNA polymerase subunit G or something similar 4a 4b 4c 4d 4e