1. Copyright OpenHelix. No use or reproduction without express written consent1

2. BLAST Sequence Similarity Searching with BLAST at NCBI Materials prepared by: Cynthia Perreault-Micale, Ph.D. Donna Messersmith, Ph.D. www.openhelix.com Updated: Q1 2011 Version 3.0_0510

3. Copyright OpenHelix. No use or reproduction without express written consent3 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

4. Copyright OpenHelix. No use or reproduction without express written consent4 BLAST: a Sequence Alignment Workhorse! “Of all the sequence alignment algorithms, the one that is most widely used is BLAST (basic local alignment search tool). It is typically used to compare one query nucleotide or protein sequence against a database of sequences, and uncover similarities and sequence matches. Its success and popularity comes from its combination of speed, sensitivity, and statistical assessment of the results.” (http://www.stanford.edu/dept/itss/docs/oracle/10g/datamine.101/b10698/10blast.htm)

5. Copyright OpenHelix. No use or reproduction without express written consent5 Alignment Algorithms: Global vs. Local GLOBAL LOCAL aligned from first to last, but results in many gaps smaller, more local blocks of maximized alignment Sequence Alignment Matrix exact match mis- match gap Your query In database BLAST: Basic Local Alignment Search Tool

6. Copyright OpenHelix. No use or reproduction without express written consent6 Introduction to BLAST BLAST was developed in 1990 to compare biological sequences Original BLAST publication PROTEIN 1 PROTEIN 2 BLAST most similar to Identify Unknown Proteins Suggest Possible Functions Clues to Structure or Family Membership Access to Specialized Data J Mol Biol. 1990 Oct 5;215(3):403-10

7. Copyright OpenHelix. No use or reproduction without express written consent7 BLAST is Part of NCBI’s Entrez Network  Access BLAST resource from NCBI homepage http://www.ncbi.nlm.nih.gov/ Click here Or click here

8. Copyright OpenHelix. No use or reproduction without express written consent8 Homepage Introductory information Select a genome if you like Basic BLAST Programs Specialized BLAST programs http://blast.ncbi.nlm.nih.gov Archives, Domains Expression, SNPs Align 2 sequences My NCBI “News” and “Tip of the Day”

9. Copyright OpenHelix. No use or reproduction without express written consent9 BLAST Help is Available BLAST glossary

10. Copyright OpenHelix. No use or reproduction without express written consent10 The BLAST Glossary & NCBI’s Education Link Many BLAST resources available

11. Copyright OpenHelix. No use or reproduction without express written consent11 BLAST References

12. Copyright OpenHelix. No use or reproduction without express written consent12 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

13. Copyright OpenHelix. No use or reproduction without express written consent13 BLAST Homepage: http://blast.ncbi.nlm.nih.gov Go directly to the BLAST homepage Choose your search type nucleotide protein translations http://blast.ncbi.nlm.nih.gov

14. Copyright OpenHelix. No use or reproduction without express written consent14 BLAST Program Selection Guide  BLAST Program Selection Guide in the Help section http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=FAQ#Queuetime  Our basic search will be a nucleotide BLAST or BLASTn Minimum suggested

15. Copyright OpenHelix. No use or reproduction without express written consent15 Basic Search - Query Form Select nucleotide BLAST from homepage Query page will open For more info click on “?” Leave default settings, except change database to “others”, (default is human genomic) Click on BLAST Upload files or add title Leave default Click on BLAST Enter sequence or ID NM_017617.3 Change

16. Copyright OpenHelix. No use or reproduction without express written consent16 Basic Search - in Progress! Screen appearance while search is in progress “Request ID”, or “RID”, is a unique identifier for each BLAST search You could click on the “Formatting options” link The results screen will automatically appear next

17. Copyright OpenHelix. No use or reproduction without express written consent17 Basic Search Results Multiple hits retrieved Results can be very large Top section: general information, color-coded graphical display, many links and tabular data Next: individual alignments We will step through each section of these results next Individual alignments shown General information, graphical display, links, tabular data

18. Copyright OpenHelix. No use or reproduction without express written consent18 Search Results - General Info & Options at Top

19. Copyright OpenHelix. No use or reproduction without express written consent19 Search Results - Graphical Display of Hits A color-coded graphical display of the BLAST hits Red lines: sequence alignment scores of > or = to 200, higher score indicates better alignment Pink lines: sequence alignment scores of 80-200 Mouse over to see the accession number listed in the box above the color-coded display

20. Copyright OpenHelix. No use or reproduction without express written consent20 Search Results in an Informative Table Accession numbers linked to the GenBank records Description of gene and species Sequence alignment scores Click on column headers to sort columns differently Let’s look closer at sequence alignment scores Columns can be sorted by clicking on headers Sequence alignment scores

21. Copyright OpenHelix. No use or reproduction without express written consent21 Understanding Sequence Alignment Scores In Help section “The Statistics of Sequence Similarity Scores” Many detailed explanations of statistics used

22. Copyright OpenHelix. No use or reproduction without express written consent22 Sequence Alignment Score Help in Glossary The glossary in the Help section provides explanations More basic explanations of statistics here

23. Copyright OpenHelix. No use or reproduction without express written consent23 Search Results - Links Available links for each record, more regularly added Our records have links to NCBI’s UniGene Resource (U), Gene Expression Omnibus (E) & Entrez Gene (G)

24. Copyright OpenHelix. No use or reproduction without express written consent24 Basic Search - Alignments An alignment from our search Basic info, statistics, links on top of alignment Query above, subject below, numbering to match Exact matches indicated with vertical line Gap indicates subject is missing a “T” Select sequences to retrieve from Entrez Nucleotide & see results tree Your query Found in database Top of page Some sequences may have regions with less similarity Note statistics Mismatch

25. Copyright OpenHelix. No use or reproduction without express written consent25 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

26. Copyright OpenHelix. No use or reproduction without express written consent26 How to Revisit Past Search Results Click on “Recent Results” tab from the homepage This will keep your list of results from the past 36 hours Enter your Request ID (RID) number to pull up your results Alternatively, you may register for “My NCBI” Enter RID number and click “Go”

27. Copyright OpenHelix. No use or reproduction without express written consent27 “My NCBI” Use in many Entrez databases to customize searches In BLAST it allows you to save your searches Great to reformat or alter initial search parameters Registration is free & easy

28. Copyright OpenHelix. No use or reproduction without express written consent28 Log into “My NCBI” and run search Select “Save Search Strategies” from the top of results page Using “My NCBI” to Save Your Searches Enter title to help remember

29. Copyright OpenHelix. No use or reproduction without express written consent29 Retrieving Saved Searches From “My NCBI” Now just go to “Saved Strategies” to find your previous searches An option to upload a search strategy Utilize the “view” & “download” links Select the red “x” to delete them Go to “Saved Strategies” to find your searches

30. Copyright OpenHelix. No use or reproduction without express written consent30 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

31. Copyright OpenHelix. No use or reproduction without express written consent31 Format Options – Access at Top of Pages Many formatting options you can choose Our example - from the Alignment View menu We will select “Pairwise with dots for identities” Click “Reformat” to see your results Select your new format and click here to see it

32. Copyright OpenHelix. No use or reproduction without express written consent32 Newly Formatted Results: Display with Dots Dots now indicate sequence identities Differences are now easy to spot in red

33. Copyright OpenHelix. No use or reproduction without express written consent33 Edit & Resubmit You can now change anything you want & resubmit We will add title & change database & algorithm Select dissimilar sequences Click to Resubmit Enter title Database Our original query - NM_017617.3 Select More options

34. Copyright OpenHelix. No use or reproduction without express written consent34 Same Sequence, New Database & Algorithm There are limitless options available here for you to try Title

35. Copyright OpenHelix. No use or reproduction without express written consent35 Setting Query Limits by Organism Limit your searches from the format page “Limit results” For this example we will limit to Homo sapiens Click on “Reformat” to see the new limited results Limit your search to a particular organism here

36. Copyright OpenHelix. No use or reproduction without express written consent36 Search Results Limited to Homo sapiens Can be a very quick and useful option Human only

37. Copyright OpenHelix. No use or reproduction without express written consent37 Setting Limits by Entrez Query Previously: set organism limit from format page Now we will limit in Query form with “Entrez Query” See “?” to learn more about Entrez Queries Boolean Operators Length Properties Molecular Weight

38. Copyright OpenHelix. No use or reproduction without express written consent38 Constructing Entrez Queries  Boolean Operators: AND, NOT & OR must be capitalized  Use quotes for a phrase (otherwise AND will be assumed)  Wild-card by * (asterisk), for ex: musc* finds muscle, musculature, but also muscarinic  Author search format: Cohen JK  From the bottom of page on previous slide: You can find “Writing Advanced Search Statements” at: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=helpentrez&part=EntrezHelp#EntrezHelp.Writing_Advanced_Sea

39. Copyright OpenHelix. No use or reproduction without express written consent39 Using an Entrez Query and a Protein Sequence Open protein blast link from homepage Enter sequence or ID, title & Entrez query term Leave database & algorithm as default Click on BLAST Leave as default Click on BLAST Enter sequence or ID EAW88241.1

40. Copyright OpenHelix. No use or reproduction without express written consent40 Results of BLASTp Search General Information Graphical Summary Descriptions of Hits Individual Alignments

41. Copyright OpenHelix. No use or reproduction without express written consent41 Results of BLASTp Search – a Few Details Search limits shown CDD Domains Your sequence Domains

42. Copyright OpenHelix. No use or reproduction without express written consent42 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

43. Copyright OpenHelix. No use or reproduction without express written consent43 Specialized BLAST Tools http://blast.ncbi.nlm.nih.gov Specialized BLAST Tools

44. Copyright OpenHelix. No use or reproduction without express written consent44 Aligning 2 or More Sequences Check here Compared to “Subject”: Click on BLAST Human calmodulin ID CAA36839.1 Scallop calmodulin ID P02595.2

45. Copyright OpenHelix. No use or reproduction without express written consent45 Results Similar format Similar options

46. Copyright OpenHelix. No use or reproduction without express written consent46 Search for SNPs Homepage lower left Enter sequence or ID NM_017617.3 NM_017617.3 Program Database By chromosome More options

47. Copyright OpenHelix. No use or reproduction without express written consent47 SNP Search Results – Reference SNPs To Reference SNP, or rs records Scroll down to alignments in “Pairwise with Identity” format

48. Copyright OpenHelix. No use or reproduction without express written consent48 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

49. Copyright OpenHelix. No use or reproduction without express written consent49 BLAST: Basic Local Alignment Search Tool My NCBI Search select genome BLAST Programs for: nucleotide alignments protein alignments alignments of translations Specialized BLAST programs http://blast.ncbi.nlm.nih.gov

50. Copyright OpenHelix. No use or reproduction without express written consent50 BLAST: Many Search & Output Options! statistical reports graphical displays individual alignments

51. Copyright OpenHelix. No use or reproduction without express written consent51 BLAST Agenda Introduction & Credits Basic Search “My NCBI” & BLAST Query & Display Options Specialized BLAST Tools Summary Exercises BLAST: http://blast.ncbi.nlm.nih.govhttp://blast.ncbi.nlm.nih.gov

52. Copyright OpenHelix. No use or reproduction without express written consent52