HOST. Flat- Faced Bat Horse Human Lab Large Cat Mule Muskrat Panda Pika Plateau Pika Raccoon Dog Reassortant Sea Mammal Sloth Swine Unknown Weasel

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1 Section A. search and sequence annotation exploration Upon completion of this exercise, you will be able to search for virus genome or gene/protein sequences and view detailed sequence annotations in IRD/ViPR. I. Search for influenza virus segment sequences in IRD 1. Search for sequences using structured search interfaces For this exercise, you will search for 2009 H1N1 pandemic HA nucleotide sequences. a. Go to the IRD homepage ( mouse-over Search Data in the gray navigation bar, then Search s and click Nucleotide s to load the Search page. b. You will notice you have many options to search: sequence type (nucleotide or protein), strain, virus type, segment number, host attributes, etc. Segment/nucleotide and influenza A are preselected by default. Type H1N1 in the subtype box, select 4 HA in Select Segments and Swine from Host, and enter 1999 in the From box of Data Range. c. Note that IRD shows an instant count of search results above the search criteria to help you search quickly and efficiently. When you select search criteria on search pages, you will instantly know how many records match your search criteria without clicking the Search button and actually running the search. If there are too many or not enough search results, you can quickly adjust the search criteria on the search page to better fit your needs. d. If you want to find strains with high similarity to 2009 ph1n1 sequences, click the radio button next to Include only ph1n1 sequences below 2009 ph1n1 s. Click Search to run the search. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Nucleotide Search Nucleotide Search Search for influenza sequences, proteins, and strains using two types of searches. Use the advanced search to allow you to refine your search with the more fine grained search, and you can pick your viewing options. Results matching your criteria: 386 DATA TO RETURN SELECT SEGMENTS HOST GEOGRAPHIC GROUPING Segment / Nucleotide Protein Strain VIRUS TYPE A B C SUB TYPE H1N1 * Use comma to separate multiple entries. Ex: H1N1, H7,. 1 PB2 2 PB1/PB1- F2 3 PA/PA- X 4 HA 5 NP 6 NA 7 M1/M2 8 NS1/NS2 COMPLETE SEQUENCES Include Partial s Complete Segments Only Flat- Faced Bat Horse Human Lab Large Cat Mule Muskrat Panda Pika Plateau Pika Raccoon Dog Reassortant Sea Mammal Sloth Swine Unknown Weasel Africa Antarctica Asia Europe North America Oceania COUNTRY Afghanistan Algeria Angola Antarctica Argentina Australia Austria STRAIN NAME Include near-complete sequences (IVR) * Use comma to separate multiple entries. Ex: A/chicken/Israel/1055/2008, A/chicken/Laos/16/2008. Complete Genomes Only 2009 ph1n1 SEQUENCES (SOP) Include ph1n1 sequences Include only ph1n1 sequences Exclude all ph1n1 sequences DATE RANGE From: 2009 To: YYYY To add month to search, see e. The search result will be displayed in a table as shown below. Each column is sortable by clicking the header. Now click the Flu Season header to sort records by flu season. Note: You can do advanced sorting by clicking the Display Settings button located above the result table. Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 1

2 About Us Community Announcements Links Resources Support Workbench Sign In SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Nucleotide Search Results Nucleotide Search Results Your Selected Items: 0 items selected Add to Working Set Save Search Run Analysis Download Your search returned 387 segments. Search Criteria Displaying About Us Community 50 records Announcements per page, sorted Links by Flu Resources Season in Support Workbench Display Sign Settings In Select all 387 segments f. On the Search Results References page, click on View to view the details of a segment. i. What s the name of the strain? ii. How long is the Subtype: segment? ascending order Next > Page: 1 of 8 More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA columns displayed by using the Display Settings link above. Home Nucleotide Search Results Segment Details (A/SW/SK/12-71/2009 Seg. 4) Segment Protein Complete Segment Name Accession Genome Length Subtype * Collection Influenza Segment/Protein Details Date Flu Host Species Country State/Province Season (SOP) Strain Name Download 4 HA Generate HQ PDF YesAdd Working 1701 Set H1N1 Identify 08/26/2009 Similar s Swine (BLAST) USA Minnesota Send Comments to Curator A/swine/Minnesota/074A/2009 to Segment: 4 JF HA JF No 1701 H1N1 09/28/2009 Swine Canada A/swine/MB/33/2009(H1N1) Strain Information Segment Information Primer Probe Features SNP Details Annotation References Data Sources Protein: HA Hemagglutinin Protein Information Protein Features Isoelectric Pt/molecular Weight Pfam Domain Pfam Motifs Predicted Epitopes Gene Ontologies Database Cross Strain Information*1 Complete Genome Set: Organism Name: Strain Name: 2009 Pandemic H1N1-like (SOP)?: Yes Influenza A Virus A/SW/SK/12-71/2009(H1N1) View Strain Details H1N1 Mixed and Segments Host: Swine Collection Date: 07/20/2009 Flu Season (SOP): Isolation Country: Canada Isolate: NCFAD-SK #71 GenBank Submission Date: 08/09/2011 NCBI Taxon ID: GenBank Notes: lineage: swl passaged in embryonated chicken eggs iii. Does the sequence have a perfect match with the GenBank commonly Header Notes: GenBank used Accession Numbers diagnosis JF PCR primer set? JF represent sequences from the 8 segments of Influenza A virus (A/SW/SK Hint: Look at the PCR Primer & Probe details. /12-71/2009(H1N1)). Segment Information*1 Genbank Source JF Accession: Definition: Influenza A virus (A/SW/SK/12-71/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. Authors: Nfon,C.K., Berhane,Y., Hisanaga,T., Zhang,S., Handel,K., Kehler,H., Labrecque,O., Lewis,N.S., Vincent,A.L., Copps,J., Alexandersen,S. and Pasick,J.,Nfon,C.K., Berhane,Y., Hisanaga,T., Zhang,S., Handel,K., Kehler,H., Labrecque,O., Lewis,N., Vincent,A.L., Copps,J., Alexandersen,S. and Pasick,J. Segment Number: 4 Segment Length: Pandemic H1N1-like (SOP)? Yes : View and design PCR primers PCR Primer & Probe Details The following primer/probe sets might be useful for diagnosing this segment. Click a link below to view alignment of this sequence to the primer/probe sequences and amplification details. VT-1 indicates a perfect match between this segment sequence and the primer/probe set. Genome Variant Amplicon Test Target/Specificity Assay Id Lab Test Name Segment Reference Type size (bp) (from literature) Targeted VT-1 Influenza WHO_CDC_swine Influenza 4 HA 116 bp Swine H1 influenza virus Reference A_RT-PCR_assay_ID8 A_hemagglutinin subtype 1_pandemic H1N1 test iv. Can you find SNP the (SOP) HMM/Pfam domain information? SNP Details SNP Type v. Any predicted epitopes for this segment? # SNPs mismatch 305 vi. Scroll up to the Segment Information section. Click View and design PCR primers to retrieve the sequence. vii. On the next page, a PCR Primer Design button is located above the sequence box. Click the button to get to the PCR Primer Design page. g. Click the Results breadcrumb at the top of the page to return to the results page. Here you can: i. select records and run an analysis on the selected records by mousing-over the Run Analysis button and clicking a desired analysis option. 2 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

3 ii. store selected sequences as a working set in the Workbench so that you can run various analyses on the working set. iii. download sequences in various formats by selecting sequences and then clicking the Download button. iv. save the search query to your Workbench by clicking the Save Search button. A Save Searches lightbox will pop up. You can choose to receive alerts on newly released data that match your search criteria by checking the Subscribe box. 2. BLAST for similar sequences The IRD BLAST tool utilizes the NCBI BLAST program set and has a collection of custom influenza sequence databases to search against. a. Go to the IRD site ( mouse-over Analyze & Visualize in the grey navigation bar and click Identify Similar s (BLAST) to load the BLAST page. b. Select a format of sequences provided and a database to search. c. Input sequences from one the following ways: Use sequences found in the IRD database as BLAST input sequences Upload a FASTA-formatted sequence file Paste FASTA-formatted sequences Use a working set consisting of a group of sequences saved to your Workbench SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA Home Identify Similar s (BLAST) Identify Similar s (BLAST) Compare sequences you provide or select from the IRD database consisting of selected sets of sequences from the IRD database or create your own database for comparison from a working set on your workbench. Note: An asterisk (*) = required field FORMAT OF SEQUENCES PROVIDED Blastn (nucleotide) Blastp (protein) Blastx (search protein using nucleotide translated in 6 reading frames) SELECT DATABASE TO SEARCH * Use generated database Influenza genes Influenza nucleotide sequences Nucleotides for segment 1 PB2 Nucleotides for segment 2 PB1 Nucleotides for segment 3 PA N l id f 4 H1 INPUT SEQUENCES * s can also be selected from search results or a working set in your workbench. Upload a file containing my sequences in FASTA format. Paste sequences in FASTA or raw format. Use working sets OUTPUT FORMAT Remove data from genetically manipulated strains from the result Number of Results to Display for each Input ADVANCED OPTIONS Show Select Advanced Option Select An Advanced Option OTHER PARAMETERS Strand Both(Default) Top Bottom Apply Low Complexity Filter True False Perform ungapped alignment Gap Costs Existence: 2 Extension: 4 Existence: 0 Extension: 4 Existence: 3 Extension: 3 Existence: 6 Extension: 2 Existence: 5 Extension: 2 Remove Add Another Advanced Option Clear Run d. Adjust BLAST parameters in the Advanced Options. Click the Run button. Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 3

4 e. A BLAST ticket number will be generated for the process, you can either wait till the process to finish or copy the ticket number and use it to retrieve the search later on by clicking the Retrieve an Analysis option under the Analyze & Visualize tab and then entering the ticket number. f. On the BLAST report page, all the nearest hits are listed in the top table. Click a hit to view its detailed alignment. Click an IRD link to view the hit s segment/protein details page in IRD. You can select hits by checking the corresponding checkboxes and click the Add to Working Set About Us Community Announcements Links Resources Support Workbench Sign In button located above the table to save the hits to a working set for further analysis. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA Home Nucleotide... Results Identify Similar s (BLAST) Results BLAST Report BLASTN [Sep ] Reference: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: Query: gb:fj Organism:Influenza#A#virus#A/California/04/2009 Seg Database: Influenza nucleotide sequences Seg4 57,773 sequences; 78,124,080 total letters Save Analysis Add to Working Set Id header Bit Score E Value >ird Country:China Influenza A virus (A/Hangzhou/04/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb HM >ird Country:USA Influenza A virus (A/Pennsylvania/09/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ >ird Country:USA Influenza A virus (A/California/04/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ Identify short peptides in proteins >ird Country:Japan Influenza A virus (A/Fukuoka-C/3/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete IRD provides a Short Peptide Search tool for finding cds. gb GQ short amino acid strings such as epitopes, ligand >ird Country:Japan Influenza A virus (A/Fukuoka-C/2/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete binding sites, sequence domains, etc. in target influenza cds. gb GQ334330proteins >ird Country:USA Influenza A virus (A/California/07/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb FJ a. Go to the IRD site ( mouse-over Analyze & Visualize in the gray >ird Country:USA Influenza A virus (A/California/08/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb FJ navigation bar and click >ird Identify Country:USA Influenza Short A virus (A/California/07/2009(H1N1)) Peptides segment in Proteins 4 hemagglutinin (HA) gene, to complete load cds. gb FJ the search 3364 page. 0.0 b. Three search types are provided: >ird Country:USA Influenza A virus (A/California/04/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb FJ >ird Country:LAB Influenza A virus (A/California/04/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb JF >ird Country:USA Influenza A virus (A/California/08/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ >ird Country:Japan Influenza A virus (A/Kagoshima/1/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ >ird Country:Japan Influenza A virus (A/Fukuoka-C/1/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ >ird Country:USA Influenza A virus (A/California/04/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete EXACT Match finds only exact matches of the query string; cds. gb GQ >ird Country:USA Influenza A virus (A/California/07/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb FJ FUZZY Match finds amino acid strings with over 50% identity to the query string; >ird Country:USA Influenza A virus (A/California/07/2009(H1N1)) hemagglutinin (HA) gene, complete cds. gb CY >ird Country:LAB Influenza A virus (A/reassortant/NIBRG-121(California/07/2009 x PR8)(H1N1)) segment 4 hemagglutinin (HA) PATTERN Match finds amino acid strings with 100% pairwise identity to the input gene, complete cds. gb GQ >ird Country:China Influenza A virus (A/Beijing/16/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb HQ pattern, e.g. [LIT]-x(3)-[LT]-[NQK]-x-{G}. hemagglutinin (HA) gene, complete cds. gb GQ For this exercise, we will search for proteins containing an epitope sequence ATVAGSL, so select >ird Country:LAB Influenza A virus (A/reassortant/NYMC X-179A(California/07/2009 x NYMC X-157)(H1N1)) segment sequence. gb CY the EXACT Match option and type ATVAGSL in the String to Find box. c. Input sequences from one the following ways: cds. gb HM Upload a FASTA-formatted sequence file Paste FASTA-formatted sequences Use a working set consisting of a group of sequences saved to your Workbench Select a pre-compiled IRD database For this example, select Influenza HA Hemagglutinin proteins. d. After you have selected your criteria, click Run to perform the query >ird Country:United Kingdom Influenza A virus (A/England/201/2009(H1N1)) segment 4 sequence. gb CY >ird Country:China Influenza A virus (A/Hangzhou/10/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb HM >ird Country:China Influenza A virus (A/Hangzhou/06/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb HM >ird Country:Italy Influenza A virus (A/Italy/05/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb GQ >ird Country:LAB Influenza A virus (A/reassortant/NYMC X-179A(California/07/2009 x NYMC X-157)(H1N1)) segment >ird Country:Canada Influenza A virus (A/Canada/GFA0402/2009(H1N1)) segment 4 sequence. gb CY >ird Country:Taiwan Influenza A virus (A/Taiwan/90262/2011(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb JN >ird Country:France Influenza A virus (A/Lyon/969/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete cds. gb JF >ird Country:United Kingdom Influenza A virus (A/England/399/2009(H1N1)) segment 4 hemagglutinin (HA) gene, complete e. On the results page, all HA proteins harboring the input string are listed in the table. Click View to view the hit s segment/protein details page in IRD. 4 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

5 f. You can select hits by checking the corresponding checkboxes and click the Add to Working Set button located above the table to save the hits to a working set for further analysis. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Identify Short Peptides in Proteins Results Identify Short Peptides Result Show Search Criteria Your Selected Items: 0 items selected Save Analysis Add to Working Set Download Select all 458 results Influenza A Virus A/USSR/90/77 Influenza A Virus A/USSR/90/77 Influenza A Virus A/USSR/90/77 Influenza A Virus A/USSR/90/77 Strain Name Protein Name Segment Subtype Score (SOP) Range HA Hemagglutinin HA Hemagglutinin HA Hemagglutinin HA Hemagglutinin 4 H2N H2N H2N H2N Next > Page: 1 of 10 Matched ATVAGSL ATVAGSL ATVAGSL ATVAGSL 4. Search for immune epitopes IRD imports experimentally determined immune epitope data from the Immune Epitope Database (IEDB, and integrates such data with existing protein sequence data. IRD also generates predicted MHC Class I epitopes from influenza amino acid sequences using the NetCTL algorithm. a. To search for immune epitopes in IRD, mouse-over the Search Data tab in the grey navigation bar and click on the Immune Epitopes option. b. On the Immune Epitope Search page, you can search for experimentally determined epitopes or predicted NetCTL epitopes. For this exercise, we will search for experimentally determined epitopes, so make sure the Experimentally Determined Epitopes option is selected. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Immune Epitope Search Immune Epitope Search Search for experimentally determined and predicted epitopes. Experimentally determined epitopes are obtained from the Immune Epitope Database (IEDB ). Use the "Immune Recognition Context" box to search for positive and negative epitopes. An epitope is considered positive if at least one assay result is found positive. An epitope is defined as negative if all of the assay results are negative. Click on the "More" drop down to refine your search. For details about how to use the Immune Recognition Context filter click here. Results matching your criteria: 406 ANTIGEN SOURCE Virus Type A B C Subtype: Example:, H5N1 Separate multiple entries with commas. EPITOPE TYPE Experimentally Determined Epitopes Predicted NetCTL Epitopes IMMUNE RECOGNITION CONTEXT Assay Assay Result Type Category Strain Name: Example: A/Memphis/1/1971 Example for flu type A search: A/% Separate multiple entries with commas. Use % as wildcard. B-cell T-cell MHC MORE Protein(s): PA, Segment 3 PA- X, Segment 3 HA, Segment 4 NP, Segment 5 NA, Segment 6 Epitope : Example: gkicn, GDPHCD IEDB ID: Example: Separate multiple entries with commas. HOST Chicken Chimpanzee Cow Dog Ferret Goat Human Humanized (transgene) Monkey Public Database Identifier: Example: ABH05852 Separate multiple entries with c. Next, you have the options of searching by subtype, strain name, protein, epitope sequence, IEDB ID, other public database ID, assay type, assay result, host, and more. Now we will search for Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 5

6 positive B cell epitopes in the HA protein: choose HA in the Protein(s) box; select B-cell in the Immune Recognition Context section; click Search to run the search. d. The search results page returns a list of epitopes matching your search criteria. Click View next to the third record (IEDB ID: 2790) to view the Epitope Details page. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Immune Epitope Search Results Experimentally Determined Epitope Search Results Your Selected Items: 0 items selected Add to Working Set Save Search Download Your search returned 406 epitopes. Search Criteria Displaying 10 records per page, sorted by IEDB ID in ascending Display Settings order. Select all 406 epitopes Next > Page: 1 of 41 More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of columns displayed by using the Display Settings link above. IEDB ID Proteins Epitope Matching Subtype Protein Names Host Assay Type Category Assay Result MHC ele Name MHC ele Class 570 ACKRGPGSGFFS RLN HA unknown (found 6) Human -Low -Low -High 1 Class II, ele Undetermined H-2-Dd Class II, ele Undetermined Class II, ele Undetermined II I II II Radio Imm Radio Imm Radio Imm Radio Imm Radio Imm Survival A Radio Imm 3H-Thymi 51 Chrom 3H-Thymi Any Metho 2136 AIYHTENAYVSV VSSHYNR 1 H1N1 HA Mass Spe 2790 ALNNRFQIKGVE LKS 9 HA -High Class II, ele Undetermined Class I, ele Undetermined Class I, ele Undetermined Class I, ele Undetermined Class II, ele Undetermined H-2-D Class II II I I I II II Enzyme-L 3H-Thymi 51 Chrom 51 Chrom 51 Chrom Any Metho ELISPOT 3H-Thymi e. This will display information about the epitope, a summary of assay results as well as protein structure(s) containing the epitope. Was this epitope also tested by T cell assay? SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA Home Immune Epitope Search Results Experimentally Determined Epitope Details (2790) Experimentally Determined Epitope Details Epitope Information IEDB ID : 2790 Epitope Chemical Type : Peptide from protein Epitope : ALNNRFQIKGVELKS Proteins : HA Hemagglutinin Assay Assay Type Assay Type Assay Group MHC ele MHC ele MHC ele MHC ele Assay Reference Category Class Source Source Response Species Species Taxonomy ID 3H-thymidine cell proliferation - N/A - - N/A - Mus musculus - N/A - C J Fitzmaurice1996 ELISPOT cytokine release IFNg - N/A - - N/A - Mus musculus - N/A - Sherry R C57BL/6 Crowe2006 Georgia 51 chromium release cytotoxicity I Class I, allele Mus musculus 1 undetermined BALB/c Deliyannis2002 Georgia any method enhancement of T cell II Class II, allele Mus musculus 1 response undetermined BALB/c Deliyannis2002 Georgia 51 chromium release cytotoxicity I Class I, allele Mus musculus 1 undetermined BALB/c Deliyannis2002 Georgia challenge assay decreased infectious - N/A - - N/A - Mus musculus - N/A - agent number BALB/c Deliyannis2002 K Sadler1999 3H-thymidine cell proliferation II Class II, allele Mus musculus 1 undetermined BALB/c Enzyme-Linked Immuno Detection of Ab/Ag - N/A - - N/A - Mus musculus - N/A - S Ghosh1999 Sorbent Assay (ELISA) binding BALB/c 3H-thymidine cell proliferation II H-2-d class II Mus musculus S Ghosh1999 BALB/c High 3H-thymidine cell proliferation II Class II, allele Mus musculus 1 D C Jackson1997 undetermined BALB/c Protein Structure PDB ID Protein UniProt ID Description Strain Subtype Protein Accession View Structure 1QU1 HA P03437 crystal structure of eha2 (23-185) A/Aichi/2/1968 BAF CAA AAA More View consensus sequence across all influenza A virus strains in IRD IRD provides pre-computed consensus sequence and polymorphism score at each position for all influenza A strains. IRD s Variation Analysis tool allows you to calculate polymorphism of IRD sequences or your own sequences. 6 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

7 For this exercise, you will search for the consensus and all sequence variations existing in all GenBank influenza type A sequences based on host of isolation, flu subtype and segment. a. Go to the IRD site ( mouse-over Analyze & Visualize in the gray navigation bar and click Analyze Variation (SNP). b. On the Analyze Variation (SNP) page, select Pre-computed analysis using sequences in the IRD database for a specified host, segment, and subtype in search type. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA Home Analyze Variation Analyze Variation (SNP) The IRD team has grouped sequences by segment number, flu type, subtype, and host and aligned the sequences. A consensus sequence has been determined for each group and the variation from that value has been determined for each position in the sequence. This has been done for both nucleotide and amino acid sequences. The IRD tool can perform the same analysis using nucleotide or amino acid sequences that you either select from the IRD database or upload on this page. SEARCH TYPE Precomputed analysis using sequences in the IRD database for a specified host, segment, and subtype New analysis of sequences you select from the IRD database or upload To study sequence polymorphism across different influenza A strains, all influenza type A sequences were downloaded from GenBank. These sequences were processed through the IRD curation pipeline. During this processing, pre-aligned sequences were generated with the ClustalW multiple alignment tool. Aligned sequences from the same host and segment (and subtype) were used to determine sequence polymorphism. A consensus sequence for each subtype was created by following the majority rule. For each position in the nucleotide multiple alignment, a score was generated by using a formula modified from the one as described in Crooks et al.. The score ranges from 0 (no polymorphism) to 200 (highest polymorphism). For more details on the score and general polymorphism approach, reference our Help document. SEARCH CRITERIA HOST SUB TYPE ANALYSIS SEGMENT/PROTEIN Human H1N1 Genomic Analysis 1 Clear Run c. Next, select desired host, subtype, analysis type (genomic or protein sequence), and segment/protein to analyze. Click Run. d. The analysis result page will be loaded, which shows the polymorphism score, consensus, and counts for each different base/amino acid at each position. The conservation score ranges from 0 (no polymorphism) to 200 (highest polymorphism). At each position, the consensus is the allele with frequency greater than 50%. If no allele exceeds 50%, N (for nucleotide) or Xaa (for amino acid) is used to indicate ambiguity. About Uspolymorphism Community Announcements Links Resources plot, Supportconsensus Sign Out sequence, and raw alignment are available for download. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA Home Analyze Variation Results Variance Analysis for Human H1N1, Segment 4 You are logged in as yun.zhang@jcvi.org Figure View Protein Analysis (4 HA) FASTA Format Raw Alignment Position Coding Score Consensus A G T C Deletion # s 1 no 72 A no 70 G no 73 C no 12 A no 5 A no 6 A no 66 A no 69 G no 66 C no 67 A no 7 G no 5 G II. Search for Hepatitis C virus sequences in ViPR 13 no 6 G no 69 G no 6 A no 27 A For this exercise, you will 17 search no for 3 full-length A 2250HCV 1 genome 3 sequences 3 0 using 2257 keyword search. Note 18 no 66 A no 158 N that ViPR also provides BLAST and Identify Short Peptides in Proteins for sequence-based 20 no 8 A sequence search. 21 no 2 A no 2 A no 69 A no 138 G no 69 C a. From the ViPR homepage ( click Flaviviridae to get to the family page. 26 no 3 A no 69 A no 69 C b. -over the Search 29 Data no 123tab and A click 2872 Genomes 6 1 to 1224load 368 the Genome 4471 Search page. 30 no 7 A no 38 A no 45 A no 0 A no 0 T no 0 G no 19 A Created by the ViPR/IRD 37 no team 1and licensed A under a Creative 2 Commons 4 1 Attribution-ShareAlike Unported License 7 38 no 84 G no 2 G no 73 C no 7 A no 1 A

8 c. You will notice you have many options to search: virus taxonomy, collection year, sample location, host, genome sequence length, etc. Select the following search criteria and click the orange Search button to execute the search. As you add search criteria, you will notice the number of matching hits above the light blue search box dynamically change. From within the taxonomy tree, click the plus sign next to Hepacivirus, next Hepatitis C virus, then Select next to Genotype 2. Please note that once Hepatitis C virus is selected, an HCV Virus Metadata panel will be displayed. Only some HCV sequence records have associated clinical metadata, so queries based on these metadata fields only retrieve sequences for which those fields are defined. Complete Genome: ý Complete Genome Only Geographic Grouping: ý Asia Advanced Options: ý Remove Duplicate s Country: ý China SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA VIRUS FAMILIES HELP Workbench Sign I ViPR Home Flaviviridae Home Genome Search Genome Search Search for virus genomic sequences and related information. You can search for the whole virus family or search for specified genus, species etc. You can also find your strain or genome record if you have its information, such as strain name, accession. Genome searches for Dengue virus or Hepatitis C virus can be augmented with clinical metadata criteria. Selecting the appropriate nodes in the taxonomy browser (Flavivirus, Dengue virus, Hepacivirus, Hepatitis C virus) will add metadata search panels and enable you to include these criteria. Some sequences have more metadata fields defined than others. Queries based on metadata only retrieve sequences for which those fields are defined. Results matching your criteria: 11 SELECT VIRUS(ES) TO INCLUDE IN SEARCH Jump to strain in taxonomy: COLLECTION YEAR GEOGRAPHIC GROUPING HOST SELECTION Start to type strain to get suggestions Deselect (5284/ strains selected) (2 Species complete genomes) Species: Hepatitis C virus Select (5284/ strains selected) (8 Genotypes complete genomes) Genotype: 1 Select (0/61659 strains selected) (15 Subtypes complete genomes) Genotype: 2 Deselect (5284/5284 strains selected) (27 Subtypes complete genomes) Genotype: 3 Select (0/10450 strains selected) (15 Subtypes - 64 complete genomes) COMPLETE GENOME Complete Genome Only Start: YYYY End: YYYY To add month to search, see Advance Search Options: Month Range Africa Asia Europe North America Oceania South America COUNTRY Azerbaijan Bangladesh Bhutan Brunei Cambodia China Cyprus Alpaca American Flamingo Arachnid Avian Bat Bearded Parrotbill Bird Bison Black Howler Blackbird Blue Jay Blue Tit Boar Bongo Brown- Headed Cowbird Buffalo Camel Cardinal SEARCH WITH HCV VIRUS METADATA HOST ATTRIBUTES SAMPLE ATTRIBUTES Host Gender Sample Source Male Female ascitic fluid blood cell supernatant plasma VIRUS ATTRIBUTES Virus Type Infection Type 1 1? 1a 1b 1a/2a chronic infection experimental ADVANCED OPTIONS Show Tip: To select multiple or deselect, Ctrl-click (Windows) or Cmd-click (Mac Select Advanced Option Remove Duplicate Genome s REMOVE DUPLICATE GENOME SEQUENCES Remove Duplicate s - If two or more genomes are exactly the same length and sequence, only the earliest will be returned. Rem d. The search result will be displayed in a table as shown below. Each column is sortable by clicking the header. Now click the Country header to sort records by country. Note: You can do advanced sorting by clicking the Display Settings button located above the result table. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA VIRUS FAMILIES HELP Workbench Sign In ViPR Home Flaviviridae Home Genome Search Results Genome Search Result Your Selected Items: 0 items selected Add to Working Set Save Search Run Analysis Download Your search returned 11 genomes. Search Criteria Displaying 50 records per page, sorted by Species Name, Strain Name, GenBank Accession in ascending order. Display Settings Select all 11 genomes Strain Name Species Name GenBank Accession Length Collection Date Host GenBank Host Country Mol Type PR63-2a Hepatitis C virus KF Human Homo sapiens China genomic RNA PR63cc-2a Hepatitis C virus KF Unknown China genomic RNA 8 WYHCV283-2a Hepatitis C virus HQ Human Homo sapiens China genomic RNA Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

9 e. On the Search Result page, click View next to a strain of interest to view the Strain Details. i. What s the name of the strain? What subtype is it? ii. In the Genome section, right-click View Nucleotide to open the sequence in a new window. Click the FASTA Download button above the sequence to download the sequence file to your computer. Strain Details for Hepatitis C virus Strain PR63-2a Strain Information Send Comments to Curator Strain Name: Organism: Taxonomy: Subtype: GenBank Host: Host: Isolation Country: PR63 Hepatitis C virus Flaviviridae -> Hepacivirus -> Hepatitis C virus -> Type 2a 2a Homo sapiens Human China Genome: KF GenBank Definition: Authors: GenBank Accession: Hepatitis C virus subtype 2a isolate PR63, complete genome. Lu,J., Xiang,Y., Tao,W., Li,Q., Wang,N., Gao,Y., Xiang,X., Xie,Q. and Zhong,J.,Wang,N., Lu,J., Tao,W., Xiang,X., Xie,Q. and Zhong,J. KF Length: 9607 Status: : Complete View Nucleotide and design PCR primers Number of Proteins: 11 Organism Name: GenBank Note: Mol Type: GenBank Host: Host: Isolation Country: Hepatitis C virus subtype 2a subtype: 2a genomic RNA Homo sapiens Human China Genome Image Map Hide Show Protein Information (SOP) More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of columns displayed by using the Display Settings link above. Gene Symbol Protein Product Name ViPR Locus ID CDS Start CDS End NCBI Gene ID Locus Name GenBank HCV polyprotein ViPR-generated C core protein C E1 E1 protein E E2 E2 protein E N/Ap7 p7 protein p NS2 NS2 protein NS NS3 NS3 protease/helicase NS NS4a NS4A protein NS4a NS4b NS4B protein NS4b NS5a NS5A protein NS5a NS5b NS5B RNA-dependent RNA polymerase NS5b iii. View the genome image map. Click E2 in the image map or View next to E2 in the Protein Information table to display the details of the protein. Look at the Genomic Annotation section. How long is the CDS? iv. Click the epitope count in the Protein Features section. How many epitopes have been found on E2? Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 9

10 III. Search for Human Herpesvirus 1 sequences in ViPR 1. Search for glycoprotein D protein sequences in Human Herpesvirus 1 a. From the ViPR homepage ( click Herpesviridae to get to the family homepage. b. -over Search Data in the grey navigation bar and click Genes & Proteins. c. The Gene/Protein Search page allows you to search for sequences based on taxonomy, collection year, sample location, host selection, gene symbol, gene product name, genomic location, ortholog group, etc. A dynamic number of matching search results is displayed at the top of the page to help you search more efficiently. Select the following criteria and click the orange Search button to run the query. Taxonomy: Human herpesvirus 1 (Herpesviridae -> Alphaherpesvirinae -> Simplexvirus -> Human Herpesvirus 1). Complete Genome Only: uncheck Gene Symbol: US6 d. The Search Results page will be displayed. How many sequences did you find? e. Now try a search based on ViPR ortholog clusters. Either: Return to the Search page by clicking Gene/Protein Search in the breadcrumb, OR Click the Search Criteria button located at the top of the search results table. f. Select the following criteria and click the orange Search button to run the query. Taxonomy: Human herpesvirus 1 (Herpesviridae -> Alphaherpesvirinae -> Simplexvirus -> Human Herpesvirus 1). Complete Genome Only: uncheck Ortholog Group: US6 10 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

11 Gene/Protein Search Search for virus protein/gene and related information. You can search for the whole virus family or search for specified genus, species etc. You can also find your strain or genome record if you have its information, such as strain name, accession. Results matching your criteria: 261 SELECT VIRUS(ES) TO INCLUDE IN SEARCH Jump to strain in taxonomy: COLLECTION YEAR GEOGRAPHIC GROUPING HOST SELECTION Start to type strain to get suggestions Deselect Species: Human herpesvirus 1 Deselect (6371/6371 strains selected) (6371 Strains - 15 complete genomes) Species: Human herpesvirus 2 Select (0/6784 strains selected) (6784 Strains - 7 complete genomes) Species: Leporid herpesvirus 4 Select (0/3 strains selected) (3 Strains - 1 complete genomes) Species: Macacine herpesvirus 1 Select (0/48 strains selected) (48 Strains - 2 complete genomes) Species: Macropodid herpesvirus 1 Select (0/5 strains selected) (5 Strains - 0 complete genomes) COMPLETE GENOME Complete Genome Only Start: YYYY End: YYYY To add month to search, see Advance Search Options: Month Range Africa Asia Europe North America Oceania South America COUNTRY Argentina Australia Austria Bangladesh Barbados Belgium Botswana Anatidae Antelope Ass Avian Babirusa Baboon Badger Bat Bear Bison Boar Bovine Buffalo Buzzard Cat Cattle Chicken Chimpanzee SEARCH TYPE Include Polyproteins in Results (SOP) Gene Symbol Gene Product Name GENOMIC LOCATION Start: End: ORTHOLOG GROUP Group Name: US6 e.g. US6 DNA polymerase ADVANCED OPTIONS Show Tip: To select multiple or deselect, Ctrl-click (Windows) or Cmd-click (MacOS) Clear Search g. The Search Results page will be displayed. How many sequences did you get this time? Note that some records do not have Gene Symbol/Gene Product Name assigned. Searching by ViPR-clustered Ortholog Group retrieves all US6 orthologs regardless of the presence of a standard Gene Symbol/Gene Product Name. h. On the Search Results page, you can: i. Save the search query to your Workbench and rerun the search again later. ii. iii. iv. Download the sequences (CDS, protein) by clicking Download. Select records and run an analysis on the selected records by mousing-over the Run Analysis button and clicking a desired analysis option. Store selected sequences as a working set in the Workbench so that you can run various analyses on the working set. v. View the details of a gene/protein. In this exercise, find Strain F, UniProt Accession number D3YPD0; then click View for this record. vi. On the Gene/Protein Details page, you will find strain information, protein information, genomic annotation, isoelectric point/molecular weight, HMM/Pfam domains, related protein structures, predicted and experimentally determined immune epitopes, GO annotations, nearest blastp hits, etc. Protein Information *2 Protein Name: envelope glycoprotein D UniProtKB Accession: D3YPD0 GenBank Protein Accession: ADD GenBank Protein GI: Protein : View Keywords: Viral envelope protein;; Virion Genomic Annotation *1 CDS CDS Start CDS End CDS Length (nt) Protein Length (aa) Codon Start View and Design Primers Source envelope glycoprotein D CDS Protein GenBank Isoelectric Point/Molecular Weight (SOP) Isoelectric pt Molecular Weight Evidence Code RCA HMM/Pfam Domains (SOP) Accession Name Description Start End PF01537 Herpes_glycop_D Herpesvirus glycoprotein D/GG/GX domain Other Domains/Motifs (SOP) Domain/Motif Start End Program transmembrane tmhmm low_complexity seg Predicted Epitopes (SOP) Prediction Details MHC Supertype # Predictions 14 A3 A2 11 A24 16 B7 17 B44 10 Total 68 Experimentally Determined Epitopes *3 Top This protein has 92 experimentally determined epitope(s). Experimentally Determined Epitope Summary Gene Ontology Classification Name GO ID Annotation Source Evidence Similar s Cellular Component integral to membrane GO: UniProtKB IEA viral envelope GO: UniProtKB IEA blastp: Swiss-Prot Searches (SOP) View Full Report Accession % Sim % S-Length % Q-Length Description E-value Q05059 SP : % % % Envelope glycoprotein D 0.0 Human herpes simplex virus 1 SP : A1Z0Q % % % Envelope glycoprotein D 0.0 Human herpes simplex virus 1 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 11

12 2. View Human Herpesvirus 1 genome annotations in GBrowse a. From the previous Gene/Protein Details page, scroll up and find the Strain Information section. Then click View Strain Details in the Strain Name row. b. The Strain Details page will be displayed. Strain Details for Human herpesvirus 1 Strain F Send Comments to Curator Genomes: Genome: Gu Genome: Af Genome: M74421 Genome: Aj Genome: Gq Genome: Gu Genome: Aj Genome: L09242 Genome: Af Genome: Ay Genome: Af Genome: Gu Genome: Gu Genome: Gu Genome: S76381 Genome: Gq Genome: U70038 Genome: U70040 Genome: U70039 Genome: Ef Strain Information Strain Name: F Organism: Human herpesvirus 1 Taxonomy: GenBank Host: Host: Isolation Country: Collection Date: Herpesviridae -> Alphaherpesvirinae -> Simplexvirus -> Human herpesvirus 1 Homo sapiens Ambiguous - see individual genome records below Ambiguous - see individual genome records below Ambiguous - see individual genome records below Genome: GU GenBank Definition: Authors: GenBank Accession: Annotations: Human herpesvirus 1 strain F, complete genome. Szpara,M.L., Parsons,L. and Enquist,L.W. GU View in GBrowse Length: Status: : Complete View Nucleotide and design PCR primers Number of Proteins: 77 Organism Name: Human herpesvirus 1 GenBank Note: Mol Type: Host: parental virus stock acronym: HHV-1;; acronym: HSV-1 genomic DNA Unknown c. Strain F has multiple genome sequences and all of the information has been integrated on one page. In the GU section, click View in GBrowse to display the genome browser. d. The GBrowse window will be displayed as shown below. The Overview panel displays the entire genome. The Region panel displays a portion of the genome surrounding the region of interest. The Details panel displays several tracks with each track corresponding to a different type of genomic feature. e. In the Protein-coding genes track, hover over a gene to view its brief description. Then click the gene to view its details. The gene/protein details page will be open in a new window. f. Enter coordinate numbers (GU734771:136, ,000) in the Landmark or Region box and click Search to view this region. What genes are in this region? g. In addition to the ViPR annotation tracks, you can display additional annotations on the genome by uploading custom tracks. The file format of a custom track can be: BED, Feature File Format, GFF, GFF3, Wiggle, BAM or SAM. Detailed information about the accepted file formats is available at: i. For this exercise, we are going to upload a polymorphism track of gd protein based on the Variation Analysis result. Download the track from: ii. iii. Click the Custom Tracks tab. Then click Add custom tracks From a file, find the track you just downloaded and click Upload. Now click the Browser tab, the uploaded track is shown in the Details panel. 12 Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License

13 h. Zoom into a 2 kbp region by selecting Show 2 kbp from the Scroll/Zoom drop-down menu. i. Scroll left or right using the < or > buttons. j. Flip the orientation by selecting the Flip checkbox such that the minus strand points to the right. This is sometimes useful for looking at minus strand genes. k. Click the ruler to show the current base position. l. Review the SNP:gD track together with the Protein-coding genes track and the IEDB epitope track. Zoom in to region Can you find B-cell epitopes on the glycoprotein D protein that are conserved across strains? m. Find and click epitope located around This will open the Epitope and Protein Affiliation Details page. Write down its amino acid positions on the gd protein ADD Experimentally Determined Epitope and Protein Affiliation Details Epitope Information IEDB ID : Epitope Chemical Type : Epitope : Proteins : Peptide from protein GFLMHAPAFETAGTY US6 envelope glycoprotein D glycoprotein D NCBI Protein Accession: ADD60053 AA Starting Position : 175 AA Ending Position: 189 Assay Assay Type Category Assay Type Assay Group Assay Response Assay Host MHC ele Class MHC ele MHC ele Source Species MHC ele Source Species Taxonomy ID Reference Enzyme-Linked Immuno Detection of Ab/Ag Mus - N/A - - N/A - Human - N/A - H J Sorbent Assay (ELISA) binding musculus herpesvirus 1 Geerligs1990 BALB/c n. You can export the genome annotations/images in multiple formats: PNG image, SVG image, GFF annotation table, or FASTA sequence file. To do so, mouse-over the File drop-down menu, then the Export as item and click a desired format to download the region you are looking at with all the features that are displayed in the GBrowse window. Created by the ViPR/IRD team and licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License 13