VIRUS PATHOGEN RESOURCE & INFLUENZA RESEARCH DATABASE EXERCISES

Transcription

1 VIRUS PATHOGEN RESOURCE & INFLUENZA RESEARCH DATABASE EXERCISES Table of Contents Exercise 1. Getting familiar with the ViPR site 1 Exercise 2. Searching sequences and exploring genome annotations 2 Exercise 3. Annotating your own genome sequence Exercise 4. Conducting comparative genomics analysis 7 Exercise 5. Analyzing Sequence Feature Variant Types (SFVT) 13 Exercise. Visualizing 3D Protein Structures 15 Exercise 7. Searching and Visualizing Influenza Virus Avian Surveillance Data 17 Exercise 1. Getting familiar with the ViPR site Upon completion of this exercise, you will be able to navigate the ViPR site, find virus families, view basic information about these groups of viruses, and know how to contact the ViPR team with questions, suggestions, or problems. a. Go to using any Internet browser (e.g. Firefox, Safari, Internet Explorer). b. The ViPR site has each virus family separated from the others, so you need to select a virus family before proceeding to search and analysis. Select a virus family that you work with. You will be taken to the virus family home page. c. On the virus family home page, you will notice a grey navigation bar consisting of the following tabs: Search Data, Analyze & Visualize, Workbench, Virus Families, and Home. These tabs are consistent across the ViPR site and are designed to help you navigate the site. d. Scroll down the page and click the Information about the virus family below the Data Summary section. e. In the blue banner, pull down the Resources menu and you can view the virus family s About page, other virus pathogen resources, anti-viral drug information, and immunology resources. f. Pull down the Support menu and view how to contact the ViPR team when you have questions, suggestions, or problems. g. Pull down the Announcements menu and view ViPR Newsletters. h. Migrate back to the ViPR homepage by clicking the ViPR logo or the Home tab. Now select Herpesviridae to get to the Herpesviridae page. 1

2 Exercise 2. Searching sequences and exploring genome annotations Upon completion of this exercise, you will be able to search for virus genome or gene/protein sequences and view detailed sequence annotations in ViPR. 2.1 Search for human herpes simplex 1 virus genome sequences in ViPR ( a. From the ViPR homepage, click Herpesviridae to get to the family homepage. b. Mouse-over Search Data in the grey navigation bar and click Genomes to load the Genomes search page. c. From within the taxonomy tree, click Select All next to Human herpesvirus 1.!"#$%"&"()*+& L$-,7B%D+,%<",*/%2$+S"7%/$3*$7$/%-E%,$.-#$E%"D+,S-#"+N%`+*%7-%/$-,7B%D+,%#B$%QB+.$%<",*/%D-S".F%+,%/$-,7B%D+,%/0$7"D"$E%2$*/>%/0$7"$/%$#7N%`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```` ^E: % ```` )+%-EE%S+#B%#+ /$-,7B>%/$$ HE<-7$%L$-,7B a0#"+/:%u+#b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d. ViPR shows a dynamic number of matching search results above the light blue search box to help you search more efficiently. If there are too many or not enough search results, you can quickly change the search criteria on the search page without clicking the Search button and running the search. e. ViPR offers a quick way to filter out partially sequenced genomes. By default, the Exclude partially sequenced genomes option is selected. If you want to include partial genome sequences in your search results, remove the checkmark. Click the Search button to run the search. Note: You also have the options of searching by collection year, sample location, host, and additional search fields in the Advanced Options section. For this exercise, we will not use these search options. f. The search result will be displayed in a table. Each column is sortable by clicking the header. Now click the Sequence Length header to sort records by length. Note: You can do advanced sorting by clicking the Display Settings button located above the result table. 2

3 !"#$%"( ((!"#$%"& X8>94*+.#/$&%0)+ 12)$&3*#4*05%#20,"#$&0/*&%D0"#$%&,-ED8)4>*"*/*&8)*F HIJKLMNK,PJ,Q, R8)4>*"* S%*T(2D>*8"%:*!*G2*&D* NL 12)$&3*#4*05%#20,,$D#8&9)+11S;,W$D#8&9)+1!S;, /*&8)%DB(= 12)$&3*#4*05%#20, *+,-.#(/#0$,%-+.$#((!"#$%&()*+,-.&/()(0(%-&(.1&2.(34%"%&5-%+$#-&7!"58&9&:-#;-%<"=== 2.2 View a genome detailed record $78&8)9+ *));>??@@@=<";#0#=+#,?0#?<";#/-)("3%=1+A.0"#+)-".B1C2DEFG=== 1*#4*05%#%:$*;<=>43$3*#4*05%#%&$*;<!%)4>*75%#20;<12)$& 3*#4*05%#20, 18)80$4%*&0 12)$&?*&8)*Y)$/*X$4 1%:*!38T 8//>*Z#$44%&/?*&@$&A180"+ a. On the Genome 180"+search result page, click next to strain H129 to view the Strain Details.!"#$%"( ((?*&@$&AB*C%&%"%8&+?*&@$&A!*G2*&D*=DD*00%8&+!*G2*&D*O*&/"3+!*G2*&D*!"$"20+!*G2*&D*+ (2)U*#8CV#8"*%&0+.#/$&%0)($)*+?*&@$&A(8"*+ X8>94*+ 12)$&3*#4*05%#20,"#$&0/*&%D0"#$%&,-ED8)4>*"*/*&8)*F HIJKLMNK,PJ,Q, R8)4>*"* S%*T(2D>*8"%:*!*G2*&D* NL 12)$&3*#4*05%#20, $D#8&9)+11S;,W$D#8&9)+1!S;, /*&8)%DB(=?*&8)*Y)$/*X$4 1%:* i. ii.!38t 8//>*Z#$44%&/!"#$%&()*%+"#,&"+-%.%)/"#$%&)01!2345 D&+E&F In the Genome section, click View DD?GH?DD&I>HH&J Nucleotide Sequence to retrieve the sequence. Click the -.!"#$%&()*#"+,$&# FASTA Download button above the sequence to download the FASTA file to your computer!"#$%&(/,+%0 /%1"#2&"13%4%(5"#$%&()!789: for later use. ;&!"#$<=(>44%??&#0 =@ABC D%=,E(!"#$%&(>44%??&#0 D%=,E(!"#$%&(D)0!"#$%&(J%K1%4%0 >FC...G9G...8HI7.@ A&%L(J%K1%4% 2&"&#(5"#$%&M(&N&O&$?(?$"%??P&Q14%Q $",?3,$&#,3(,""%?$M(?&+&3,"($#(%)R.(5N#?5N,$,?% "%S13,$#"T(?1O1&$(D>UU78M(O&Q?(5"#$%& 5N#?5N,$,?%(G($#(*#"+(,(N#3#%VT+%(4,5,O3%(#* Q%5N#?5N#"T3,$&S(%)R.P,35N,M(&2#32%Q(& $",?3,$&#,3("%S13,$&# View the genome image map. Click RL1 in the genome image map or next to RL1 in the Protein Information table to display the details of the RL1 protein. Look at the Genomic #++%$0 Annotation section. How long is the CDS? D%#+&4(>#$,$&#-G(( 17)7$8"$ 17)9: 7#+".% /%1"#2&"13%4%(5"#$%&()!789: 17 :GH G.C7 H8H.8I UJW!"#$%& D%=,E /%1"#2&"13%4%(5"#$%&()!789: G.8@H@ G.:H.: H8H.8I UJW!"#$%& D%=,E )?#%3%4$"&4(!#&$XF#3%413,"(Y%&SN$((ZJ[!\(( 0C#%-%.$"&.)/$ G.97 D#-%.+-8")E%&<=$.CG@:97 9,&:%.%)1#:% ]> [$N%"(U#+,&?XF#$&*?((ZJ[!\(( 7$8"$ 9: 3#L^4#+53%_&$T #F8&GD#$&H 7 GI?%S!"#<"8F 3#L^4#+53%_&$T H: I`?%S 3#L^4#+53%_&$T 8. :H?%S!"%Q&4$%Q(a5&$#5%?((ZJ[!\!"%:&.$&#)%$8&-C D&+E&F DI1)7+/%"$J/% >7 K)!"%:&.$&#C D G` >. G` >.8 G` iii. Click Prediction Details in the Predicted Epitopes section. What is the sequence and G&+H&F II?JK?II&G>II&E supertype of the first epitope listed? b. Return to the genome search result page by clicking the Results breadcrumb at the top of the page. 3

4 2.3 View an HSV1 genome using GBrowse GBrowse - an interactive genome viewer!"#$%&()*+,-.&/()(0(%-&(.1&2.(34%"%&5-%+$#-&7!"58&9&:-#;-%<"=== *));>??@@@=<";#0#=+#,?0#?<";#A)#("./-)("3%=1+B.0"2-%%"+.C=== Provides both bird s eye and detailed views of genomes and genome annotations. Available for reference sequences of pox and herpes viruses in ViPR.!"# $%&()*+,(-.)*/)01% %89%:!"; $%&()*+,(-.)*/)01%+< %89%:!"= $%&()*+,(-.)*/)01%+$ 3453 record %89%: a. From the genome search result page, click next to a strain 17 to view the Strain Details.!"= >/)01%+!"=?@ %89%: Strain 17 has three genome sequences and all of the information has been integrated on one page. In!"A $%&()*+*/)01%+!"A %89%: *));>??@@@=<";#0#=+#,?0#? the NC_00180 section, click the View in GBrowse!"#$%&()*+,-.&/()(0(%-&(.1&2.(34%"%&5-%+$#-&7!"58&9&:-#;-%<"=== link to display the3453 genome browser.!"bc D>+0/9%E*)/0/+1%F1G10)/+<H>I; %89%:!"BB D,JK%0+*/)01%+!"BB %89%: Or, you can the Analyze & Visualize tab in the bar 7%89%: and click View!"BL mouse-over M1/1)%+*/)01%+!"BL 3453 navigation 3453 N"B D/9%E./1*01)%9(+/,J(90)/+<H>I %89%: Genomes in GBrowse. This will return a list of all genomes that can be viewed in GBrowse. =U82"`0 =&&82&D*)*&"0 O%&A0 ]*082#!a=]R1B==!"#$%"&()*++,-+. =(=OcdaeSY!`=OYda Z.]f@a(R1 SY]`!H=XYOYa! 1.Xa =(=OcdaeSY!`=OYda 18)*?*&8)*!*$#D3 ]*02>"0!"#$%&B*"$%>0[,-\ Y:*&"%C9!%)%>$#!*G2*&D*0[@O=!\!"#$%&()"$%*+,-#./ 7%89%:+2O1%101)%P QJK9%+F/*E&1/JE+BR+.)K*(0+,%)K? =>%/&!*G2*&D*0[X!=\ 7%89%:+"SJ%.+..EE1)%P 4HTLLB=L# S%02$>%g*=>%/&*:!*G2*&D*0 "SJ%.+%%)0901)%EP M1U+1%+78/)UE!"#)"9: /*&8)*+CbJKLMNK$;$/*&8)*+&DQQ,NQP$;$/*&8)*+7,^,,M$; Y:*&"%C9!38#"V*4"%:*0%&V#8"*%&0 "SJ%.+V%,0FP B@CC#B "SJ%.+"090JEP H)K*(0?*&8)*=&&8"$"8#[?=`\ %-,3.#$#<),3-.)#$$ "SJ%.P M1U+4J.()01W+"SJ%. =&$>9g*!*G2*&D*S$#%$"%8&[!(V\ 4JKG/+)O+>/)01%EP ;;!"#$%&($)*+,X*"$:$"$!*G2*&D*=&$>90%0 X/,9%1EK+49KP QJK9%+F/*E&1/JE+B.#/$&%0)+ 12)$&3*#4*05%#20,?*&*#$"*V39>8/*&*"%D#** 1*#4*05%#%:$*;<=>43$3*#4*05%#%&$*;<!%)4>*75%#20;<12)$& Y)(+D-*P,%)K1.+24!"#$%&()*+,-.&/()(0(%-&(.1&2.(34%"%&5-%+$#-&7!"58&9&:-#;-%<"=== *));>??@@@=<";#0#=+#,?0#?,0#+@%-=1+A%-B$-.-2-%%"+.CDEF=== $78&8)9+?*&@$&A180"+ 180"+ 7%)K+<K9,+Y9*++ 3*#4*05%#20, S%*T?*&8)*0%&?@#8T0* 18)80$4%*&0 1Y!.]c ]*"#%*5*$&=&$>90%0 +Q1W "F)U+++++ D),,(+Z/9**1%,!"#)":$=>?@ABC@$$ panel displays the entire b. The GBrowse window will be displayed as shown below. The Overview 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. ]*"#%*5*$B8T&>8$: c82#=&$>90%01%0"8#9?*&@$&ab*c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`U%G2%"%&aL>%/$0*YRVQ `O, a&5*>84*/>9d84#8"*%&o `OM `#$D%>;B(=/>9D809>$0* `OL (2D>*$# #8"*%&`OL `OJ V#8U$U>*=V;:*4*&:*&"3*>%D$0*`OJ! "4!=>0.)))!?4! -2459%*+)@9;AB!!! (2D>*$#4#8"*%&`O^ `O^! QC$ `OP R$40%:48#"$>4#8"*%& `O- */2)*&"4#8"*%&`O- `O,L!*#%&*_"3#*8&%&*;4#8"*%&A%&$0*M `O,M B*879#%U8&2D>*$0* `O,, */2)*&"4#8"*%&`O,, `O,J B(=4$DA$/%&/"*#)%&$0*02U2&%", `O,- B(=4$DA$/%&/"*/2)*&"4#8"*%&`O,- `O,P */2)*&"4#8"*%&`O,P `OMQ a&5*>84*4#8"*%&`omq `O,K X$b8#D$40%:4#8"*%& * * * * * * *N9+OT:*:-,.",. D&+E&F * * * * * * *F$C-)(;U>V0U -.7.1< :0/1;C $958: 0A09A04;A!"#"$%"&$(")&*+,&-.&/ %&5+7"8(&4%&9:;<"<&=>&(3"&*$(4+;$#&?;%(4(:("&+9&@##"A>&$;<&?;9"8(4+:%&4%"$%"%&B*?C&D&CCEF&:;<"&G+;($8(&*+H&CCE*/I//00-000J1G&$;<&4%&$&8+##$=+$(4+;&="(K"";&*+(3+5 L:MM$;&C"$#(3&?2.&N;4,"%4(>&+9&2"O$%&E+:(3K"%(";&P"<48$#&G";("&$;<&Q"8;$&2"83;+#+A4"%H&Q4:%&4M$A"%&8+:("%>&+9&GG&R:=#48&C"$#(3&?M$A"&S4=$>.&T"##8+M"&?M$A"%.&NHEH "5$(M";(&+9&Q"("$;%&@99$4%&.&E84";8"&+9&(3"&?;,4%4=#"&$;<&Q4$#U+;".&EK4%%&?;%(4(:("&+9&V4+4;9+M$(48%H 4 D&+E&F 8)109$23" (*2#85%#2>*&D*4#8"*%&YRVL^FJ B$"D"%/!<DE8;!05:25=>:D=! `OK ]*4>%D$"%8&8#%/%&;U%&:%&/4#8"*%& 9:@S*G#-.",*SN/!#F%&GG--, `ON B(=3*>%D$0*_4#%)$0*D8)4>*7;$008D%$"*:4#8"*%& N"%D#$.-,/!4:8;=7:243;89!:5AD9843:!H$FG a&5*>84*/>9d84#8"*%&x I-D).-,/!,*,I,//I,) `O,Q J5K!A5;5!?5489=!;!JFL `O,^ */2)*&"4#8"*%&`O,^ * * * * * * *G#-.",RD-(,K*K","% 4#-,"5$7 ]O, GG?DH?GG&I>DJ&K

5 c. 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. d. Select a region to view: i. Enter gene symbol UL23 in the Landmark or Region box and click Search to display UL23 in the Details panel. Does it have Pfam domains? ii. Enter coordinate numbers (NC_00180:1..10,000) in the Landmark or Region box and click Search to move to this region. What genes are in this region? e. Click and drag the ruler that shows base position. f. Zoom out to a 20 kbp region by selecting Show 20 kbp from the Scroll/Zoom drop-down menu. g. Scroll left or right using the < or > buttons. h. 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. i. If you want to display additional data on the genome, for example, a region involved in virus reactivation, you can add your own tracks to the GBrowse window. Click the Custom Tracks tab. Then click From text in the Add custom tracks section. In the text box, type in NC_ reactivation and click Upload. Now click the Browser tab, the uploaded track is shown in the Details panel. j. 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. 5

6 Exercise 3. Annotating your own genome sequence Genome Annotator (GATU) is tool provided by the Influenza Research Database and the Virus Pathogen Resource to help users annotate their own genome sequences. To use GATU, you will need to select a previously annotated reference sequence and then use GATU to transfer the annotations to a target genome sequence. In this exercise, we will use HSV1 strain 17 as the reference sequence and HSV1 strain H129 genome sequence as our target sequence to be annotated. a. Mouse-over the Analyze & Visualize tab from the grey navigation bar and click Genome Annotator (GATU). b. Click Launch GATU and allow the GATU app to be loaded to your computer. c. Download HSV1 strain 17 NC_ GenBank file from NC_ d. In the GATU window, upload the strain 17.gb file as the Reference Genome and the H129 FASTA file saved from Exercise 2.2 as the Genome to Annotate. e. Click Annotate to execute annotation process. When done, a table is displayed which summarizes the similarities of transferred annotations and provides users with checkbox control over which to accept. f. Click the Save button to save the annotated target genome in multiple file formats: Genbank, EMBL, or XML.

7 Exercise 4. Conducting comparative genomics analysis Upon completion of this exercise, you will be able to: search for virus sequences and view detailed information about these sequences in ViPR, perform a multiple sequence alignment and phylogenetic tree construction on a select set of sequence records to infer their evolutionary relationships, and identify nucleotide or amino acid positions that differ significantly between groups of viruses distinguished by specific host attributes. In the following exercise, we will use Dengue virus 2 as an example to search and analyze sequences. The Dengue virus is native in tropical areas of the world and is endemic in areas where it co-localizes with the preferred Aedes aegypti mosquito vector. It can logically be assumed that DENV infections reported in clinics located in non-tropical regions of the world, and the United States, are likely due to recent travel to endemic regions by the patient. These import cases can thus establish viral lineage in new regions as a result of human travel. The CDC has previously determined that by examining the recent travel history of patients having been clinically diagnosed with DENV between 1999 and 2000 validates such an explanation (2). As an example ViPR use case, we will extend the CDC study by performing an in-depth comparative genomics analysis of all DENV 2 isolates taken between the years of 1999 and This will involve the following bioinformatics workflow: 1) identify sequence records in the ViPR database using the metadata-driven query interface; 2) save selected sequence records as a working set in a personal workbench; 3) construct a phylogenetic tree using an optimal model of evolution and color the tree to highlight metadata differences; 4) visualize multiple sequence alignments; and 5) perform a metadata-driven comparative analysis of sequence variation. Similar tasks can be performed for other viruses to address other biological questions by combining the wealth of relevant data with the suite of bioinformatics tools integrated into the ViPR resource. 4.1 Search for Dengue virus Type 2 genome sequences isolated from human a. Go the ViPR homepage and click Dengue in the Featured Viruses section to get to the Dengue virus page. b. Mouse-over the Search Data tab and click Genomes to load the Genome Search page. You will notice you have many options to search: virus attributes, host attributes, clinical attributes, etc. From within the Taxonomy tree, click Select All next to Dengue virus type 2, enter in Collection Year, select Human in Host Selection and click the orange Search button. c. 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. 7

8 d. On the Genome search result page, click to view the Strain Details. i. What s the name of the strain? Where was it isolated? ii. Is there any clinical metadata associated with this strain? iii. View the genome image map. Click E in the image map or next to E in the Protein Information table to display the details of the Envelope protein. Look at the Genomic Annotation section. How long is the CDS? iv. Can you find the HMM/Pfam Domains information? v. Click Prediction Details in the Predicted Epitopes section. What is the sequence and supertype of the first epitope listed? e. Click the Results breadcrumb at the top of the page to return to the Genome search result page. You can select records and run analysis on the selected records by mousing-over the Run Analysis button and clicking the desired analysis option. In the next exercise, we are going to store the sequences as a working set in the Workbench feature so that we can run various analyses on the working set. 4.2 Register for a Workbench and store sequences as a working set for further analysis The ViPR Workbench provides free online storage space for you to: Save and organize working sets of sequences, analysis results and search criteria Share working sets and analysis results with others Upload personal sequences and combine with existing working sets a. Select all records by checking the checkbox above the table and then click the Add to Working Set button. Now you need to register for a Workbench account in order to use this feature. b. In the pop-up lightbox, you will be prompted to log in or register for a new account. If you don t have a Workbench account yet, choose the Register for a new account button, enter your , password of your choice, name and institution, and click Register to complete the registration process. Alternatively, you can click either the Register for a Workbench link under the Workbench tab or the Workbench Sign In link in the top-right corner to get to the Workbench Registration page. 8

9 c. Enter a name and description for the working set. After the working set is saved to your Workbench, you will be able to view and run analysis on the working set during subsequent visits. d. Click the Workbench tab from the grey navigation bar to go to your Workbench. You ll see the saved working set as well as unsaved searches and analyses you just ran during this session. e. Click next to the working set that you just saved to display items in the working set. Now you will see the analysis tools available for your data type under the Run Analysis drop-down menu. Move on to the next exercise. 4.3 Multiple sequence alignment ViPR uses two algorithms to align sequences: For most viruses that ViPR supports, MUSCLE is used to align sequences. For Poxviridae and Herpesviridae virus genome sequences, Mauve is used for multiple sequence alignment. Mauve is a powerful alignment algorithm for long sequences and genomes with rearranged sequences and horizontal transfer blocks. a. Select all genomes in the working set by checking the checkbox above the table. Then mouse-over Run Analysis and click Align Sequences (MSA). b. Click Continue using default Nucleic Acid (Genome) option. c. On the next page, select FASTA or any other desired output format and click Run. d. Once the analysis finishes, mouse-over the blue Run Analysis button and click Visualize Aligned Sequences. 9

10 e. On the Visualize Aligned Sequences Customization page, you can customize the sequence label in the alignment by selecting the Custom radio button and selecting one or more options from the list, e.g. Strain Name and Country. Click Run to load the visualized alignment. f. Customize sequence labels in your alignment: On the Visualize Aligned Sequences page, right-click a strain name in the alignment, mouse-over the sequence name in pop-up menu, click Edit Name/Description, modify the name as desired, and click Accept. g. Highlight a sequence region on your alignment: Within the JalView alignment visualization window, click and drag a desired region of sequence alignment, right-click the selected region, mouse-over Selection and click Create Sequence Feature. h. Color alignment based on sequence identity cutoff: Examine various options to adjust the alignment display (e.g. File, Format, etc.). Click Colour pulldown menu and then the Above Identity Threshold option. Using sliding bar, adjust color display such that only residues with >80% sequence identity are colored. Scroll left and right to view results. 10

11 4.4 Build a phylogenetic tree a. Create a phylogenetic tree from the aligned sequences using the Quick Tree model. On the Visualize Aligned Sequences page, click the Generate Phylogenetic Tree button. b. On the next page, select Quick Tree and click Build Tree. The analysis will take a couple of minutes to run. You can save the analysis to your Workbench by entering a name and then clicking Save to Workbench. Move to other parts of the exercises, and then come back to the Workbench to retrieve the analysis results. c. After the analysis is completed, click View Tree to open the tree window. In the Tree Decorations section of the tree viewer window, click the dropdown menu below Basic Decoration Options and choose country. Click Show Legend to display the color code for different countries. d. Now, color tree leaves by the Advanced Decoration. In the Advanced Decorations section, click the drop-down menu and choose Region. You will see that the tree is now colored by continents: Asia, North America, and South America. e. Change the decoration colors. Click the Advanced Decoration again. In the pop-up Advanced Decoration Options window, click Manual Decoration then click Go to change the color. Check North America and choose red in the color palette, then click Apply. f. Run tree model compare to determine which model fits your data best. Return to the Generate Phylogenetic Tree page using the breadcrumb feature. Choose the Custom Tree model and then Run ModelCompare tool for recommendation of evolutionary model that best fits my data. Which model of evolution fits best? 4.5 Metadata-driven Comparative Analysis Tool for Sequences (Meta-CATS) Metadata-driven Comparative Analysis Tool for Sequences A unique comparative genomics analysis tool in ViPR to identify nucleotide /amino acid positions that significantly differ between two or more groups of virus sequences. For this exercise, we are going to compare the genome sequences from the previous phylogenetic tree. a. Click the Workbench tab, find the working set you just saved and click to display the working set. Or click the breadcrumb of Working Set at the top of the page. 11

12 b. Select all sequences from the working set. Then mouse-over the Run Analysis tab and click Metadata-driven Comparative Analysis Tool. c. We are going to separate these sequences into two groups by the two clades from the phylogenetic tree analysis excluding the Brazil strain and the Viet Nam strain and use 0.05 as our significance cutoff value, so keep the default settings on the page and click Continue. d. Now, assign the sequences into two groups by double-clicking the strains from the top clade (DENV_2/US/BID_1428/1999, DENV_2/US/BID_1425/1999, strains from other countries except Brazil and Viet Nam) in the top box and clicking the Add link above either of the boxes at the bottom of the page. Repeat the process to assign all the other US strains and the Brazil strain to the other group and click the Run button. e. This analysis may take a few minutes to finish. You can save the analysis to your Workbench and retrieve it later. To do so, enter in a name and click Save to Workbench. O\3;M(13<3 )LAO(a3XL!L\O MfX\ d"+(#*0(4"0$(%&(#,(5+&:h/#&i+.,"+./s0,.0*&:0$!"#$%$%&%#( M"U0( (X0.#$#.#_1*%V0&(;"U=#*#.%V0(T0&"U%7,(3&#45,%,!"#$%$#$&()*"+,-./0$($#)*",1"+./)23,4+$53)3,7..5,&,8"#90,89:3"#, 1"+240(74%7[("&(./0(T0&"U0,(."(,0407.(./0UF(./0&(#$$(./0U(."(./0%*(*"+=,(25(74%7[("&(c3$$c:(c0U"V0c(S%44(,0&$(./0(,0407.0$(T0&"U0,(2#7[(."(./0(U#%&(4%,.:(d"+(7#&(#4,"($*#./0(0&"U0,(."(./0%*(*"+=,: )*+,-.+/0-12-/3453,$3/ T0&2#&[(#770,,%"&B\A]PPDR^(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DRG]NDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\A]EPGGG(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DR]DNGHHH(Z(1#.0BGHHH(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\AREGPGQ(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)`QENDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\A]PPDRR(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DRGPNDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\A]EPGG^(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DR]GNGHHH(Z(1#.0BGHHH(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\AREGP^H(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)`QQNDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#&.+/0-12-/3453,$3/-! $$(Z(0U"V0 T0&2#&[(#770,,%"&B\A]PPDRG(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DRG`NDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&B\A]PPDR`(Z(O.*#%&(&#U0B1\K)_GNAON>L1_)DRGENDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BAO3(Z(M",.BM+U#& T0&2#&[(#770,,%"&BaCPRRPR`(Z(O.*#%&(&#U0B1\K)_GNKLN>L1_)G^]GNGHHH(Z(1#.0BGHHH(Z(;"+&.*5BK%7#*#+#(Z(M",.BM+U#& T0&2#&[(#770,,%"&BaCE`HH]`(Z(O.*#%&(&#U0B1\K)_GNKLN>L1_)G]]RNGHHH(Z(1#.0BGHHH(Z(;"+&.*5BK%7#*#+#(Z(M",.BM+U#& T0&2#&[(#770,,%"&BaCEQERPP(Z(O.*#%&(&#U0B1\K)_GNKLN>L1_)GQG^NGHHH(Z(1#.0BGHHH(Z(;"+&.*5BK%7#*#+#(Z(M",.BM+U#& T0&2#&[(#770,,%"&BTbDQQEQ`(Z(O.*#%&(&#U0B1\K)_GNKLN>L1_)G]E^NDQQQ(Z(1#.0BDQQQ(Z(;"+&.*5BK%7#*#+#(Z(M",.BM+U#&.+/0-12-/3453,$3/-! $$(Z(0U"V0 $%&(!"# f. When the analysis completes, two tables will be displayed on the page. The first gives the statistical results for individual positions, while the second table reports the statistical results for pairs of groups found to contribute to the significant result at each column. g. Click the blue Show P-Values Bar Plot button to view the p-values for the positions identified as significantly different between your specified groups. h. Go back to the same working set, generate and view a new multiple sequence alignment. Find the positions of the significant nucleotide differences. 12

13 Exercise 5. Analyzing Sequence Feature Variant Types (SFVT) At the end of this exercise you will be able to identify regions of viral proteins with known structural, functional and immune epitope properties, and will be able to assess the level of sequence variation in these regions. Sequence Feature Variant Type (SFVT) can help you identify sequence variations that may correlate with phenotypic characteristics, e.g., drug sensitivity/resistance, virulence, transmissibility, etc. Sequence Features (SFs): specific regions defined based on functional properties, structural properties, and immune epitope locations. Obtained from literature and/or imported from other databases and validated by domain experts. Variant Types (VTs): polymorphisms in each Sequence Feature are identified as Variant Types of the Sequence Feature (SFVT). The SFVT analysis is available for influenza A virus in the Influenza Research Database and Dengue virus serotypes 1-4, Hepatitis C subtype 1a and Vaccinia viruses in the Virus Pathogen Resource. Note: Use the Influenza Research Database ( for this exercise. You may do SFVT analysis of Dengue, Hepatitis C 1a, or Vaccinia viruses in the Virus Pathogen Resource ( by using this workflow. a. From the grey navigation bar, mouse over Search Data and click Sequence Feature Variant Types. b. The SFVT landing page will be loaded. Here you can search or browse the Sequence Features of influenza A virus. Click Go to Sequence Feature List to browse all Sequence Features of influenza A proteins. c. Look at the Sequence Feature list of influenza A virus. How many Sequence Features are there for the NS1 protein? d. Click the number in the Functional SF category for NS1 to view all functional Sequence Features of NS1. e. Can you find the Sequence Feature that is involved in the nuclear export of NS1 protein? Write down the name of the Sequence Feature. f. Click to view details about this Sequence Feature. What are the sources of information that support its functional classification? In which strain was this sequence feature defined?!"#$"%&"(")*$+","(-%-*-.%!"#$%&()*+% ),-,%./%0%(1%*$/"%()*+% 234/%5*(7),-7/04%*"8%9:#"$8;&<*47-=>?--@,%./%0%(1%*$/"%(2A 234/%5*(7),-7,1-B CD8-(,$"*&(?"%3%"%0%(;$"*&@ EFG#"E>H?I=)H@ J%3%"%0%(!#;&$&# -=>8-K>!.$+&"!*+)-%/!0!12345!3789 E!/%"$# J&0#EBE=K :* %2;<53!12345 =1>8819!12345 )445>>819?,=31589! =2<@ "A8B5945 &1B5> &1;;59 CD8- -=>8-K> LMN==OB- 8)E8!/PQ%GRSOTN-SK UF&:"#$R!N=KST VW!,%./%0%(*GG%G($#(* X%$%"#4#<#/;(:"#$%&(0*/;%; /04%*"(%9:#"$Y(Z-KK(*G(Z-KT *"%(%;;%$&*4(3#"($X&;(*0$&[&$\Y 13

14 g. How many variant types exist for this Sequence Feature in IRD? Which reference strain defines Variant Type 1 (VT-1)? h. Click the number in the Strain Count column for VT-1 to view detailed metadata about the strains harboring this Variant Type. Select the Date column heading to re-order table by date of isolation. In what year was the earliest strain isolated? i. Click the number in the Strain Count column for VT-8 to view detailed metadata about the strains harboring this Variant Type. What do these strains have in common? Note: You can use the toolbar to download the full data set. j. Return to the previous page. Click the blue Find a VT(s) button to expand the VT search panel. Now search for Variant Types with a single mutation at I145. Change the I145 to? to search for any residue at this position, i.e., amino acids IFDRLETL?LL How many Variant Types do you find that differ from the reference strain at only amino acid position 145 of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

15 Exercise. Visualizing 3D Protein Structures At the end of this exercise you will be able to find and visualize protein structures for virus proteins and to adjust the images to highlight selected protein features..1 Use the Influenza Research Database ( to examine an influenza virus protein structure. a. From the grey navigation bar, mouse over Search Data and click 3D Protein Structures. b. Search for the 3D structures of influenza A (H3N2) HA protein. Virus Type: ý A Subtype: H3N2 Select Proteins to search: ý 4 HA c. On the 3D protein structure search result page, choose the 1EO8 protein structure and click View Structure to get to the structure page. d. You ll be taken to the Jmol protein structure visualization page. Use mouse in display window to change view. In the Display Options section, change the Display Type to Secondary Structure in Cartoon to view the secondary structures of your protein. e. Highlight the structure with a sequence conservation score by selecting Sequence conservation computed using all sequences within the Highlight Sequence Conservation section. f. In the Highlight Ligands section, check Highlight Ligands in green. g. In the Highlight Epitopes section, highlight B-cell epitopes. Are there any B cell epitopes that are entirely conserved among influenza virus strains in the database? h. In the Highlight Sequence Features section, highlight in pink the following sequence feature on the structure: Influenza A_H3_sialic-acid-binding_98(19) (Influenza A_H3_ SF1). Examine relationship between amino acid residues known to be required for sialic acid binding and position of sialic acid ligands in the protein crystal structure. i. In the Highlight by Swiss-Prot Position section, highlight residues 22 and 228, which have been found to influence virus host range. 15

16 j. Rotate the structure as you need. Download the protein structure image with highlighted residues by clicking Save View As Image beneath the image..2 Use the Virus Pathogen Resource ( to examine a protein structure of human simplex 1 virus. a. Go to the Herpesviridae page in ViPR. From the grey navigation bar, mouse-over Search Data and click 3D Protein Structures. b. Search for the 3D structures of human simplex 1 virus by choosing human herpesvirus 1 from the taxonomy tree. c. On the 3D protein structure search result page, choose the 1E2I protein structure and click View Structure to get to the structure page. d. You ll be taken to the Jmol protein structure visualization page. Use mouse in display window to change view. In the Display Options section, change the Display Type to Secondary Structure in Cartoon to view the secondary structures of your protein. e. In the Highlight Ligands section, check Highlight Ligands in green. f. In the Highlight by Swiss-Prot Position section, highlight residues 18-17, which have been found to be the nucleoside-binding site. g. Rotate the structure as you need. Download the protein structure image with highlighted residues by clicking Save View As Image beneath the image. 1

17 Exercise 7. Searching and Visualizing Influenza Virus Avian Surveillance Data After this exercise you should be able to identify influenza surveillance samples from selected parts of the world, determine the proportion that are flu positive and view their geospatial relationships with known bird flyways. Wild birds are the natural reservoirs of influenza viruses. Influenza surveillance in different wild bird populations is critical for understanding the transmission and evolution of these viruses, and assists in the early detection and warning of highly pathogenic avian influenza. The Influenza Research Database (IRD) serves as a central repository of influenza surveillance data for the NIH-NIAID funded Centers of Excellence for Influenza Research and Surveillance (CEIRS) program. a. Search surveillance data from avian host collected in Africa using the Influenza Research Database ( From the grey navigation bar, mouse-over Search Data and click Animal Surveillance Data. On the next page, choose the following parameters: Surveillance Data Type: ý Avian Sample Selection: Uncheck all boxes Geographic Grouping: ý Africa Note: Use the Search Criteria button to change search criteria to answer the following questions. i. How many total African avian surveillance samples are currently found in the IRD? ii. How many have been tested for the presence of influenza virus? iii. How many samples are flu-positive? iv. How many have associated sequence records currently in IRD? b. Display surveillance records for all tested samples on a map. Adjust Search Criteria to only select all tested samples and select all records; click the orange View on Map button. c. Color code by the percent of flu-positive samples. Check the checkbox next to Show percent flupositive samples. d. Highlight bird flyways and see if there is a correlation between flyways and bird samples. Check Black sea/mediterranean. Which countries might be expected to share similar influenza virus strains based on this bird flyway? 17

18 e. Zoom in to find at least one sampling location in Egypt that has flu-positive samples. What s the name of the location (City, Province)? On the surveillance map, click to view details about the records at that location. f. View the surveillance records of flu-positive samples. i. From Step e., in the pop-up window, follow Click here to view all the records at the location. This will bring up all surveillance records collected from this location. ii. Sort records by the Positive for flu column. iii. Click next to a flu-positive sample to see the sample collection, the host, etc. References 1. Pickett, B.E., Sadat, E.L., Zhang, Y., Noronha, J., Squires, R.B., Hunt, V., Liu, M., Kumar, S., Zaremba, S., Gu, H., Zhou, L., Larson, C., Dietrich, J., Klem, E. B., Scheuermann, R. H. (2011) ViPR: An Open Bioinformatics Database and Analysis Resource for Virology Research. Nucl. Acids Res. doi: /nar/gkr CDC. (2002) From the Centers for Disease Control and Prevention. Imported dengue--united States, 1999 and MMWR Morb Mortal Wkly Rep, 51, Squires, B., Macken, C., Garcia-Sastre, A., Godbole, S., Noronha, J., Hunt, V., Chang, R., Larsen, C.N., Klem, E., Biersack, K. et al. (2008) BioHealthBase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence. Nucleic Acids Res, 3, D Squires, R.B., Noronha, J., Hunt, V., García-Sastre, A., Macken, C., Baumgarth, N., Suarez, D., Pickett, B.E., Zhang, Y., Larsen, C.N., Ramsey, A., Zhou, L., Zaremba, S., Kumar, S., Deitrich, J., Klem, E., Scheuermann, R.H. (2011) Influenza Research Database: An integrated bioinformatics resource for influenza research and surveillance. Influenza and Other Respiratory Viruses, manuscript in revision. 18