Home

Ingenuity Variant Analysis Plugin

Contents

1. 2014 QIAGEN Inc Al rights reserved Figure 6 3 Copy the link provided in one of the output files and paste it into an internet browser to go directly to the specific variant analysis on the Ingenuity Variant Analysis page 2 Open the variant track that was produced as one of the outputs Right click on the variant track in the Genome Browser View and select Launch Ingenuity Variant Analysis figure 6 4 This will also send you directly to the Ingenuity Variant Analysis web page CHAPTER 6 ANALYSIS USING THE PLUGIN AND THE IVA WEB INTERFACE 20 als normal73 DNA X 50 000 000 100 000 000 62685228 z M Launch Ingenuity Variant Analysis normal73 DNA seq trimmed paired Variant Analysis Update sarcoma 73 DNA se a a waar pea J Find in Navigation Area 7 o l li File Edit View Toolbox Show v v v v v ME oY Figure 6 4 Right clicking on the variant track in the Genome Browser View and selecting Launch Ingenuity Variant Analysis will send you directly to the specific variant analysis on the Ingenuity Variant Analysis page Ingenuity Variant Analysis enables you to apply a number of different filters The example in figure 6 5 shows a filter cascade with the default filters Variants 415 x Confidence T15 x Common Variants T14 x Predicted Deleterious B25 x Genetic Analysis 290 169 g4 W Recalculate when filters change Figure 6 5 An example of an Ingenuity Variant Analysi
2. Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or create a customized pipeline on the Ingenuity web interface e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e f the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Analysis e Analysis description choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 5 Specify the parameters for the Fixed Ploidy Variant Detection tool for the f
3. Settings MANIEE Figure 10 1 Accessing the user settings inside the Ingenuity Variant Analysis web interface 3 Change your Allele Frequency Community opt in status using the checkbox figure 10 2 Optin to share anonymized pooled statistics derived from my samples and in access to the Allele Frequency Community database Apply Figure 10 2 Changing the Allele Frequency Community opt in status using the checkbox inside user settings 49
4. Help in the Menu Bar Plugins and Resources E or Plugins 4 in the Toolbar The plugin manager has three tabs at the top e Manage Plugins This is an overview of plugins that are installed e Download Plugins This is an overview of available plugins on CLC bio s server e Manage Resources This is an overview of resources that are installed To install a plugin click the Download Plugins tab This will display an overview of the plugins that are available for download and installation see figure 1 1 Clicking a plugin will display additional information at the right side of the dialog This will also display a button Download and Install Click the Ingenuity Variant Analysis and press Download and Install A dialog displaying progress is now shown and the plugin is downloaded and installed If the Ingenuity Variant Analysis is not shown on the server and you have it on your computer e g if you have downloaded it from our web site you can install it by clicking the Install from File button at the bottom of the dialog This will open a dialog where you can browse for the plugin The plugin file should be a file of the type cpa When you close the dialog you will be asked whether you wish to restart the CLC Genomics Workbench The plugin will not be ready for use until you have restarted In order to install plugins on Windows the Workbench must be run in administrator mode Right click the program shor
5. Family information 1 C Father affected amer ain Mother Variant track for mother FFF Sample3 Variants Mother affected Previous ext nish E Cancel Figure 5 4 Specify family data for your analysis Data for at least one parent must be specified at this step Click on the button labeled Next to go to the next wizard step where you have the possibility to specify further family data for the analysis figure 5 5 Similarly to the previous step you can specify for each sibling a variant track disease status and gender Click on the button labeled Next to go to the next wizard step where you must specify your Ingenuity username email address and password figure 5 6 If you do not have an Ingenuity CHAPTER 5 INGENUITY VARIANT ANALYSIS FOR HEREDITARY DISEASES 16 Ingenuity Variant Analysis for Hereditary Diseases Family information 2 Choose where to run 2 Select variant track for Sibling 1 optional proband Variant track for sibling 1 Variant analysis Sibling 1 affected arameters ap Gender of sibling 1 Other Unknown Family information 1 Sibling 2 optional 3 Family information 2 Variant track for sibling 2 Sibling 2 affected Gender of sibling 2 Other Unknown Previous Ingenuity Variant Analysis for Hereditary Diseases Account information Choose where to run Select variant track for proband Variant analysis spas Account information Family information 1 I
6. CHAPTER 8 REMOVE VARIANTS FOUND IN ALLELE FREQUENCY COMMUNITY 28 Filtering parameters Choose where to run 2 Select variant track 3 Reference and Account information Community frequency Filtering ters s lt r Sa Maximum frequency 3 0 Figure 8 4 At this step you can specify the filter cutofff to be used by the Remove Variants found in Allele Frequency Community tool Result handling Choose where to run 2 Select variant track 3 Reference and Account information lt Filtering parameters De re Figure 8 5 The result handling step in the Remove Variants found in Allele Frequency Community wizard without being saved In this case you will have to manually save the outputs if you would like to keep them If you choose to save the outputs they will not be opened automatically but will be saved at the destination you have specified When the analysis is finished the resulting track will contain an additional column named Community Frequency containing the observed frequencies in percent of the variants in the Allele Frequency Community Furthermore the number of variants will have been reduced according to the cutoff parameter that you have specified Note that if the Community Frequency column is empty for a variant it indicates that the variant was not found in the Allele Fre
7. choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 6 Specify the parameters for the Fixed Ploidy Variant Detection tool for the father mother and proband successively figure 9 19 The parameters that can be set are e Required variant probability is the minimum probability value of the variant site required for the variant to be called Note that it is not the minimum value of the probability of the individual variant For the Fixed Ploidy Variant detector if a variant Site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold For example if the required variant probability is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 CHAPTER 9 WORKFLOWS 45 Identify and Interpret Causal Variants in Trio using IVA WES s Geni tale Fixed Ploidy Variant Detection father 1 Select reads from a Configurable Parameters 2 Select reads from mo
8. probability of the individual variant For the Fixed Ploidy Variant detector if a variant site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold For example if the required variant probability is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 e Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair reads are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters e Minimum coverage Only variants in regions covered by at least this many reads are called e Minimum count Only variants that are present in at least this many reads are called e Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called 6 On the last wizard window p
9. Analysis Variant analysis parameters 1 Choose where to run Data configuration 2 Select variant tracks Reference Homo sapiens hg19 sequence Control samples 8 3 Variant analysis parameters Analysis configuration Analysis pipeline name Personal genome w Disease name Any Cancer Custom analysis name Naming Analysis name input date Analysis Press Shift F1 for options ut date Description Analysis description Shift F1 for options an Previous gt Next Finish Cancel Figure 4 3 At this step you can specify the analysis parameters for the Ingenuity Variant Analysis tool e Reference Select the human reference sequence that is found under CLC_References in the Navigation Area Only complete human genomes can be used as references We currently Support the human reference genome hg19 The use of selected regions of a genome e g individual chromosomes is not supported e Control samples This is an optional parameter You may select one or more variant tracks which will be considered to be control samples in the analysis e Analysis pipeline name Select the pipeline appropriate for your analysis The Ingenuity Variant Analysis is performed with predefined settings that differ depending on your choice of analysis pipeline The following options are available Personal genome Useful if you have a single sample and are looking for variants with known disease or phenotypic associati
10. Database A oo My Remove False Positives Remove Germline Variants iE Remove Reference Variants pE Remove Variants Inside Genome Regions pE Remove Variants Outside Genome Regions pE Remove Variants Outside Targeted Regions 4 From Databases aa Bie Remove Variants Found in 1000 Genomes Project a pe o Remove Variants Found in Common dbSNP r Remove Variants Found in HapMap Figure 8 1 The Remove Variants found in Allele Frequency Community tool can be found in the toolbox in the folder Remove Variants From Databases If you are connected to a server you will first be asked about where you would like to run the analysis If you are not connected to a server the first step is to specify the input for the analysis The Remove Variants found in Allele Frequency Community tool accepts a single variant track FFE as input Select the desired variant track as shown in figure 8 2 Click on the button labeled Next to go to the next wizard step where you can specify your Ingenuity username password and the reference sequence as described below figure 8 3 e Ingenuity username email address used to log in to Ingenuity Variant Analysis 26 CHAPTER 8 REMOVE VARIANTS FOUND IN ALLELE FREQUENCY COMMUNITY 2 Select variant track 1 Choose where to run heise aac Sdected elements 1 2 Select variant track Ea LC Data Hek Sample1 Variants GE Data ee Demo o bbb Sample Variants l Sample3 Variants
11. Other Unknown Analysis name input date Analysis Press Shit F1 for options Anohyie deaniption input date Description Press Shit F1 for options Ingenuity VA username email address cxxx dxxx giagen com Ingenuity VA password gt Locked Settings Figure 9 11 Specify a reference and login information to Ingenuity Variant Analysis Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or create a customized pipeline on the Ingenuity web interface e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e f the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the
12. Select sequencing reads Navigation Area Selected elements 1 Select reads from sibling Select reads from father le if CLC_Data workflows Select reads from mother Trio gt Family of F Selectreads from proband E fa F Father affected E Mother unaffected Sibling unaffected Qy lt enter search term gt Previous Figure 9 2 Specify the sequencing reads for each family members successively 3 Specify the parameters for the Fixed Ploidy Variant Detection tool for the sibling figure 9 3 n Identify and Interpret Causal Variants in Family of Four using IVA WGS Fixed Ploidy Variant Detection sibling Configurable Parameters Required variant probability 50 0 1 Select reads from unaffected sibling 2 Select reads from father Ignore broken pairs v 3 Select reads from mother Mini cnerage 4 Selectreads from proband Minimum count 5 Fixed Ploidy Variant Minimum frequency Detection sibling b Locked Settings L JES Figure 9 3 Specifying the parameters for the Fixed Ploidy Variant Detection tool The parameters that can be set are CHAPTER 9 WORKFLOWS 32 Required variant probability is the minimum probability value of the variant site required for the variant to be called Note that it is not the minimum value of the probability of the individual variant For the Fixed Ploidy V
13. Targeted Amplicon Sequencing amp Hereditary Disease H Identify and Annotate Variants in a Family of Four using IVA TAS U2 1 Double click on the Identify and Annotate Variants in a Family of Four using IVA TAS tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Specify a target region file figure 9 9 This is a file that depends on the technology you used for sequencing Identify and Interpret Causal Variants in Family of Four using IVA TAS Select input for targeted region file Navigation Area Selected elements 1 Pio BG targeted_sequencing iF gt t 0293689_Regions_BED CTFR Cergentis AmpliSeq agilent_sure_select 5 4450293689_Regions_BED m 1 Select targeted region file j Qy lt enter search term gt X Cancel Figure 9 9 Specify a target region file 3 Select the sequencing reads for the sibling father and mother and proband figure 9 10 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member i Identify and Interpret Causal Variants in Family of Four using IVA TAS Select sequencing reads Navigation Area Selected elements 1 Glo CLC_Data a Affected child Select reads from
14. Variants in Trio using IVA WES PE EPSA Ingenuity Variant Analysis for Hereditary Diseases Ls See Configurable Parameters 2 Select reads from mother Reference 2 Homo sapiens hg19 sequence 3 Select targeted region Analysis pipeline name Variant Analysis Genetic Disease Pipeline file Disease inheritance pattern Other unknown Select reads from proband Custom analysis name Analysis for Hereditary Father aff j Diseases Mother affected Analysis name input date Analysis Press Shit Fl input date Description Press Shit Fl for options Analysis description Ingenuity VA username email address cxxx dxxx giagen com Ingenuity VA password gt Locked Settings Figure 9 18 Specify a reference and login information to Ingenuity Variant Analysis e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e f the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Analysis e Analysis description
15. analysis name is the name that is shown on the Ingenuity Variant Analysis page as the name when you choose My Analyses The same analysis name can furthermore be used in a Variant Analysis Custom Pipeline if you specify it in the Custom analysis name field See above e Analysis description This will be the description of the analysis in Ingenuity Variant Analysis once created There are a few shorthand notations available input will be substituted with the name of the input experiment date is substituted with a date stamp Click on the button labeled Next to go to the next wizard step where you must specify your Ingenuity username email address and password figure 4 4 If you do not have an Ingenuity username or password you must first create an Ingenuity account Account creation is described in chapter 3 Ingenuity Variant Analysis Account information 1 Choose where to run 2 Select variant tracks 3 Variant analysis parameters Account information Account information Ingenuity username email address XXxx xxxx com Ingenuity password TIT titi Previous gt Next nish Cancel Figure 4 4 Specify the account information your Ingenuity username email address and password are required at this step Click on the button labeled Next to go to the last wizard step figure 4 5 where you can set the output options If the Import annotated and filtered variants option is che
16. filter settings see figure 6 7 Choose Variant Analysis Update and save the updated variant track in the Navigation Area The updated variant track will be saved with the name extension IVA update which means that the original variant track will not be overwritten by the updated variant track In normal73 DNA X 36 normal73 DNA seq 7 Launch Ingenuity Variant Analysis trimmed paired f Variant Analysis Update i p Find in Navigation Area File F Edit View t Toolbox b Show gt HE ag Figure 6 7 If you have made changes to the used filters you can import the updated variant track into the Workbench by right clicking on the original variant track and choosing Variant Analysis Update When you have imported the updated variant track we recommend that you open the updated variant track in split view with the table view After running the variant analysis the variant table will contain additional columns holding Ingenuity Variant Analysis specific information The type of analysis performed and which filters were used will determine which of these columns see figure 6 8 will be added to your results Please visit the Ingenuity websitehttp www ingenuity com products variant analysis for more information about the wide range of options available on the Ingenuity Variant Analysis web page If you would like to learn more about Ingenuity Variant Analysis annotations please see http ingenuity force com variants Varian
17. output are generated e 3 Reads Track one for each family member 3 Filtered Variant Track one for each family member An Imported track e A URL file 9 5 Identify and Interpret Causal Variants in a Trio WES To run this workflow go to Toolbox Ready to Use Workflows Whole Exome Sequencing f or Targeted Amplicon Sequencing Hereditary Disease Identify and Annotate Variants in a Trio using IVA WES CHAPTER 9 WORKFLOWS 43 1 Double click on the Identify and Annotate Variants in a Trio using IVA WES tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Select the sequencing reads for the father and mother respectively figure 9 16 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member r Bx Identify and Interpret Causal Variants in Trio using IVA WES Select sequencing reads 1 Select reads from father ais Navigation Area 2 Select reads from mother CLC_Data workflows ty Trio 4 Affected child i Father vr E Qy zenter search term gt Figure 9 16 Specify the sequencing reads for each family member successively 3 Specify a target region file figure 9 17 This is a file that de
18. sibling E workflows Trio Selectreads from father E gt Family of Four Affected child eT nac thom moth ise ae Father affected 5 Select reads from proband E Mother unaffected E Sibling unaffected Select targeted region file Qy lt enter search term gt Previous Figure 9 10 Specify the sequencing reads for each family members successively 4 Specify your reference and parameters for the Ingenuity Variant Analysis for Hereditary Diseases as well as your login information figure 9 11 The parameters that can be set are e Reference Select the genome sequence you would like to work with usually hg19 e Analysis pipeline name specify which kind of analysis you would like to perform on your variants CHAPTER 9 WORKFLOWS 38 in Identify and Interpret Causal Variants in Family of Four using IVA TAS cs Ingenuity Variant Analysis for Hereditary Diseases L i targeted region Configurable Parameters Reference Homo sapiens hg 19 sequence Select reads from sibling A Analysis pipeline name Variant Analysis Genetic Disease Pipeline Disease inheritance pattern Other unknown Select reads from mother Custom analysis name Select reads from father Select reads from proband Gender of proband Other Unknown t Father affected Ingenuity Variant Analysis for Hereditary Mother affected Diseases Sibling 1 affected Gender of sibling 1
19. supplement the abilities of the Workbench with the biological knowledge available in Ingenuity Variant Analysis and the Allele Frequency Community The plugin bundles four tools which can be found in the toolbox e Ingenuity Variant Analysis used to analyze personal genomes cancer genomes or to Carry out stratification analysis e Ingenuity Variant Analysis for Hereditary Diseases used to analyze genetic diseases e Add Information from Allele Frequency Community used to annotate with information from the Allele Frequency Community database e Remove Variants found in Allele Frequency Community used to filter out variants that are present in the Allele Frequency Community database In addition to the four tools the plugin comes with six ready to use workflows for analysis and interpretation of hereditary diseases These workflows are installed under the respective applications in the toolbox Whole Genome Sequencing Whole Exome Sequencing or Targeted Amplicon Sequencing The six workflows are e Identify and Interpret Causal Variants in a Family of Four using IVA WGS e Identify and Interpret Causal Variants in a Trio using IVA WGS e Identify and Interpret Causal Variants in a Family of Four using IVA WES CHAPTER 3 INTRODUCTION TO THE INGENUITY VARIANT ANALYSIS PLUGIN 8 e Identify and Interpret Causal Variants in a Trio using IVA WES e Identify and Interpret Causal Variants in a Family of Four using IVA TAS e Identify and I
20. the two generated outputs will be opened in the View Area without being saved In this case you will have to manually save the outputs if you would like to keep them If you choose to save the outputs they will not be opened automatically but will be saved at the destination you have specified When the analysis is finished the resulting track will contain an additional column named Community Frequency containing the observed frequencies in percent of the variants in the Allele Frequency Community If the Community Frequency column is empty for a variant it indicates that the variant was not found in the Allele Frequency Community Chapter 8 Remove Variants Found in Allele Frequency Community The Remove Variants found in Allele Frequency Community tool allows you to add Community Frequency annotations from the Allele Frequency Community to variant tracks and to filter the variants based on those annotations To be able to obtain Community Frequency annotations from the Allele Frequency Community your Ingenuity user account must be opted in to the Allele Frequency Community Chapter 10 describes how to change your Allele Frequency Community opt in status The Remove Variants found in Allele Frequency Community tool is installed in the toolbox in the following location figure 8 1 Remove Variants From Databases Remove Variants Found in Allele Frequency Community al Toolbox b Remove Variants Not Found in External
21. z NEPA n 1 6885154 Exonic CAMTA1 p D40H i missense Damaging x Common Variants 6084 1522 t 1 8930567 SUTR Exonic ENO1 p V62I missense Damaging 1 9992027 Exonic LZIC p T146A a missense Tolerated 1 10459713 Exonic PGD p A12A m synonymous ENCODE TFBS POLR2A 26 x Predicted Deleterious i 3633 4479 n 1 10473258 Exonic PGD p K265R missense Tolerated i 11982726 Exonic KIAA2013 p C618W missense Damaging 1 11982728 Exonic KIAA2013 p C618fs 12 frameshift 2863 118 r a 11982829 Exonic KIAA2013 p A584G missense Tolerated 184 1 19923523 5 UTR MINOS1 MINC ENCODE TFBS BRCA1 CHD2 162 v 1 19923532 5 UTR MINOS1 MINC ENCODE TFBS BRCA1 CHD2 158 M Recalculate when filters change 1 20945045 Exonic CDA p L1420 missense Damaging Legend hide 1 20945056 Exonic CDA p Q146 ams stop gain Function Goniidant Cal 1 26230206 Exonic STMN1 p S38fs 17 frameshift N es Da se 1 26230302 Exonic STMN1 p l6V x missense Tolerated Identical to Refere nome 1 26607417 Exonic SH3BGRL3 _ p C71fs 5 frameshift Heterozygous Variant n i z E iid i ii Heterozygous Ambig 1 26607420 Exonic SH3BGRL3 p C71fs 20 frameshift Homozygous variant 1 271407022 Exonic ARID1A p 519945S p 52 synonymous Copy Number Gain Het y z Copy Number Gain H yg 1 28931896 Exonic TAF12 p E146E synonymous 1 me Hemizygous z TP 1 29474620 FUTR SRSF4 A Nullizy ae sa bx a Gene o 1 29474624 3UTR SRSF4 No genotype 5 Se ees ra ee eee 5 b
22. 2s CLC_References O zenter search term gt C Batch Reference and Account information 1 Choose where to run 2 Select variant track Account information 3 Reference and Account information Ingenuity username email address xxxx xxxx com Ingenuity password CO Reference information Reference 7f Homo sapiens hg19 sequence Next Next J Finish Figure 8 3 At this step you can specify the analysis parameters for the Remove Variants found in Allele Frequency Community tool e Ingenuity password password corresponding to your Ingenuity username e Reference select the human reference sequence that is found under CLC_References in the Navigation Area Only complete human genomes can be used as references We currently Support the human reference genome hg19 The use of selected regions of the genomes e g individual chromosomes is not supported Click on the button labeled Next to go to the next wizard step Here you can specify the cutoff for filtering by entering the desired value in the Maximum frequency field figure 8 4 Only variants whose Allele Frequency Community frequency is equal to or lower than the specified value will be considered Click on the button labeled Next to go to the final wizard step figure 8 5 where you can set the output options If you choose to open the results the two generated outputs will be opened in the View Area
23. D i Ww j 1 Select targeted region file 4 Q lt enter search term gt Figure 9 5 Specify a target region file 3 Select the sequencing reads for the sibling father mother and proband successively figure 9 6 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member m Identify and Interpret Causal Variants in Family of Four using IVA WES Select sequencing reads Navigation Area Selected elements 1 o CLC_Data a affected child Select reads from sibling E workflows Trio Selectreads from father 5 3 Family of Four Select reads from mother am Affected child Father affected Select reads from proband F Mother unaffected Sibling unaffected Select targeted region file vr a Q lt enter search term gt Previous gt Next Figure 9 6 Specify the sequencing reads for each family members successively 4 Specify your reference and parameters for the Ingenuity Variant Analysis for Hereditary Diseases as well as your login information figure 9 7 The parameters that can be set are e Reference Select the genome sequence you would like to work with usually hg19 e Analysis pipeline name specify whi
24. Frequency 10 Heterozygous Sample 1 ponte 15 10 J NHLBI ESP Frequency 10 Heterozygous Sample 1 Variants ee bg V NHLBI ESP African Frequency lt gt NHLBI ESP European Frequency all Track from Selecti Create Track from Selection J Ingenuity Variant Findings 3 Figure 6 1 The result of the Ingenuity Variant Analysis opened in the Genome Browser View in Biomedical Genomics Workbench The variant track is shown in split view with the variant table e A document providing a link to the Ingenuity Variant Analysis page see figure 6 2 Copying this link and pasting it into an internet browser will take you to the Ingenuity Variant Analysis page where you can narrow down your analysis further by applying different filters or by adjusting the predefined filter settings 18 CHAPTER 6 ANALYSIS USING THE PLUGIN AND THE IVA WEB INTERFACE 19 Navigation Area 4 ate j x E 23N_R1_001 p X k E Ea B O Y https api ingenuity com datastream analysisStatus _ ie IVA A gt be 23N_R1_001 paired 23T_R1_001 paired Annotated 23N_R1_001 paired 23T_R1_001 paired Annotat ES Ingenuity Variant Analysis log 4 TT s Figure 6 2 Copy this link into an internet browser to see the result of the Ingenuity Variant Analysis e A log file if you ticked the Open log box There are two different approaches to how you can handle the identified variants e Open the variants in the Genome Bro
25. P Ingenuity Variant Analysis Plugin USER MANUAL User manual for Ingenuity Variant Analysis plugin Windows Mac OS X and Linux September 7 2015 This software is for research purposes only CLC bio a QIAGEN Company Silkeborgvej 2 Prismet DK 8000 Aarhus C Denmark CC big A QIAGEN company Contents 1 Installation of the Ingenuity Variant Analysis plugin 2 Uninstall 3 Introduction to the Ingenuity Variant Analysis plugin 4 Ingenuity Variant Analysis 5 Ingenuity Variant Analysis for Hereditary Diseases 6 Analysis using the plugin and the IVA web interface 7 Add Information from Allele Frequency Community 8 Remove Variants Found in Allele Frequency Community 9 Workflows 9 1 Identify and Interpret Causal Variants in a Family of Four WGS 9 2 Identify and Interpret Causal Variants in a Family of Four WES 9 3 Identify and Interpret Causal Variants in a Family of Four TAS 9 4 Identify and Interpret Causal Variants ina Trio WGS 9 5 Identify and Interpret Causal Variants ina Trio WES 9 6 Identify and Interpret Causal Variants ina Trio TAS 222 08 10 Changing Allele Frequency Community opt in settings 13 18 23 26 30 31 33 37 40 42 45 49 Chapter 1 Installation of the Ingenuity Variant Analysis plugin The Ingenuity Variant Analysis is installed as a plugin Plugins are installed using the plugin manager
26. VA password gt Locked Settings Figure 9 7 Specify a reference and login information to Ingenuity Variant Analysis e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e f the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Analysis e Analysis description choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 5 Specify the parameters for the Fixed Ploidy Variant Detection tool for the proband mother sibling and father successively figure 9 8 The parameters that can be set are e Required variant probability is the minimum probability value of the variant site required for the variant to be call
27. Y DISEASES 17 Result handling Choose where to run 2 Select variant track for Output options Import annotated and filtered variants 1 Variant analysis ter a Result handling Family information 1 Open 5 Family information 2 C Save 6 Ace aymanon TE Result aah A Open log Figure 5 7 The result handling step in the Ingenuity Variant Analysis for Hereditary Diseases wizard If you choose to open the results the two generated outputs will be opened in the View Area without being saved In this case you will have to manually save the outputs if you would like to keep them If you choose to save the outputs they will not be opened automatically but will be saved at the destination you have specified The outputs are described in chapter 6 Note that after the analysis has been performed the filter settings used for the Ingenuity Variant Analysis can be manually adjusted How to do this is also described in chapter 6 Chapter 6 Analysis using the plugin and the IVA web interface When the analysis is complete you will get different kinds of output e A variant track with the annotated and filtered variants figure 6 1 This track can be opened in a Genome Browser View by double clicking on the name of the variant track in the Navigation Area ls Sample Vari X gt Track Settings Show more tracks together gy Create Geno
28. able Parameters Select reads from mother Reference 2 Homo sapiens hg 19 sequence Select reads from proband Analysis pipeline name Variant Analysis Genetic Disease Pipeline Fixed Ploidy Variant Disease inheritance pattern Other unknown Detection proband Custom analysis name Ingenuity Variant Gender of proband Other Unknown Analysis for Hereditary Fain wale el Diseases Mother affected Analysis name input date Analysis Press Shift Fil f Pav ca Pd input date Description Press Shift Fi f tions Ingenuity VA username email address cxxx dxxx giagen com Ingenuity VA password b Locked Settings Figure 9 15 Specify a reference and login information to Ingenuity Variant Analysis The parameters that can be set are e Reference Select the genome sequence you would like to work with usually hg19 e Analysis pipeline name specify which kind of analysis you would like to perform on your variants Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants CHAPTER 9 WORKFLOWS 42 Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or cre
29. ariant detector if a variant site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold For example if the required variant probability is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair reads are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters Minimum coverage Only variants in regions covered by at least this many reads are called Minimum count Only variants that are present in at least this many reads are called Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called 4 Specify your reference and parameters for the Ingenuity Variant Analysis for Hereditary Diseases a
30. ate a customized pipeline on the Ingenuity web interface e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e If the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Analysis e Analysis description choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 5 Specify the parameters for the Fixed Ploidy Variant Detection tool for the mother and the father as you did previously 6 On the last wizard window pressing the button Preview All Parameters allows you to preview all parameters At this step you can only view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Four types of
31. ather mother and proband respectively as you did previously 6 On the last wizard window pressing the button Preview All Parameters allows you to preview all parameters At this step you can only view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Four types of output are generated e 4 Reads Track one for each family member e 4 Filtered Variant Track one for each family member e An Imported track e A URL file 9 2 Identify and Interpret Causal Variants in a Family of Four WES To run this workflow go to Toolbox Ready to Use Workflows Whole Exome Sequencing f or Targeted Amplicon Sequencing H Hereditary Disease 3 Identify and Annotate Variants in a Family of Four using IVA WES HS CHAPTER 9 WORKFLOWS 34 1 Double click on the Identify and Annotate Variants in a Family of Four using IVA WES tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Specify a target region file figure 9 5 This is a file that depends on the technology you used for sequencing Identify and Interpret Causal Variants in Family of Four using IVA WES Select input for targeted region file Navigation Area Selected elements 1 targeted_sequencing a E 0293689_Regions_BED CTFR Cergentis AmpliSeq z iy 3 agilent_sure_select 3 F 450293689_Regions_BE
32. ch kind of analysis you would like to perform on your variants Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or create a customized pipeline on the Ingenuity web interface CHAPTER 9 WORKFLOWS 35 in Identify and Interpret Causal Variants in Family of Four using IVA WES EE Ingenuity Variant Analysis for Hereditary Diseases x i targeted region Configurable Parameters Reference 2 Homo sapiens hg 19 sequence Analysis pipeline name Variant Analysis Genetic Disease Pipeline Disease inheritance pattern Other unknown Select reads from mother Custom analysis name Select reads from sibling Select reads from father Select reads from proband Gender of proband Other Unknown Father affected Ingenuity Variant Analysis for Hereditary Mother affected Diseases Sibling 1 affected Gender of sibling 1 Other Unknown Analysis name input date Analysis Press Shit F1 for options input date Description Press Shit Fl for options Analysis description Ingenuity VA username email address cxxx dxxx giagen com Ingenuity
33. cked the tool will produce a variant track as output If it is unchecked the analysis will be created and can be accessed inside the Ingenuity Variant Analysis web interface but the results will not be imported into the workbench Note it is not possible to import results if you have selected the Upload only pipeline earlier in the wizard If you choose to open the results the two generated outputs will be opened in the View Area without being saved In this case you will have to manually save the outputs if you would like to keep them If you choose to save the outputs click on the button labeled Next to specify where to save the results and click on the button labeled Finish to start the Ingenuity Variant Analysis Your results will not be opened automatically but will be saved at the destination you have specified The outputs are described in chapter 6 Note that after the analysis has been performed the CHAPTER 4 INGENUITY VARIANT ANALYSIS 12 Result han 1 Choose where to run ding 2 Select variant tracks Output options 3 Variant analysis Import annotated and filtered variants parameters 4 Account information Result handling Open O Save Log handling _ Open log Figure 4 5 The result handling step in the Ingenuity Variant Analysis wizard filter settings used for the Ingenuity Variant Analysis can be manually adjusted How to do this is also described in chapter 6 Chapter 5 Ingenuity Variant Analysis fo
34. customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or create a customized pipeline on the Ingenuity web interface CHAPTER 9 WORKFLOWS 41 i Identify and Interpret Causal Variants in Trio using IVA TAS Ingenuity Variant Analysis for Hereditary Diseases 1 Select reads from father ConligealPaancions 2 Select reads from mother Reference 26 Homo sapiens hg 19 sequence 3 Select targeted region Analysis pipeline name Variant Analysis Genetic Disease Pipeline file Disease inheritance pattern Other unknown g Select reads from proband Custom analysis name Ingenuity Variant Gender of proband Other Unknown Analysis for Hereditary Father aff Diseases Mother affected Analysis input date Analysis Press Shift F input date Description Dress S t Fi a options Analysis description Ingenuity VA username email address cxxx dxxx giagen com Ingenuity VA password gt Locked Settings Figure 9 22 Specify a reference and login information to Ingenuity Variant Analysis e Custom analysis name Enter a name in this field only if you have selected the Variant Analysis Custom Pipeline in the field above e Gender of proband you can choose between male female ambiguous for babies born with sex chromosomes anomalies or
35. e set You can also specify command line instructions he Alignments and Trees _ HEE Create Alignment Create Pairwise Comparison Create Tree The additional alignments in the toolbox Allignment methods Three different alignment methods are included in this extension ClustalW ClustalO and Muscle For more detailed information on each of Figure 1 1 The plugins that are available for download Chapter 2 Uninstall Plugins are uninstalled using the plugin manager Help in the Menu Bar Plugins and Resources E or Plugins 4 in the Toolbar This will open the dialog shown in figure 2 1 c g Download Plugins Manage Resources r Manage Plugins and Resources p CLC bio suppor t dcbio com Version 1 5 1 Guild 131211 2142 102901 Perform alignments with ClustalO ClustalW and MUSCLE Ones Daal Annotate with GFF file Q CLC bio suppor t dcbio com Version 2 2 6 Build 131211 2143 102901 Using this plug in it is possible to annotate a sequence from list of annotations found in a GFF file Located in the Toolbox CLC Microbial Genome Finishing Module CLC bio support dcbio com Version 1 3 2 Build 140318 1029 Various tools for genome finishing aimed to dose and produce high quality genomes in sequencing projects CLC Workbench Client Plugin Q CLC bio suppor t dcbio com Version 6 0 Build 140207 0940 105889 Client plugin for connecting to a CLC Genom
36. ected regions of the genomes e g individual chromosomes is not Supported e Analysis pipeline name Select the appropriate pipeline for your analysis The Ingenuity Variant Analysis is performed with predefined settings that differ depending on your choice of analysis pipeline The following options are available Variant Analysis Genetic Disease Pipeline to be used if you are studying genetic disease 13 CHAPTER 5 INGENUITY VARIANT ANALYSIS FOR HEREDITARY DISEASES 14 Select variant track for proband Navigation Area Selected elements 1 2 Select variant track for 5 25 CLC_Data rE Sample 1 Variants proband GPG Data eS Demo BEN series Variants kk Sample Variants Hk Sample3 Variants H E CLC_References 1 Choose where to run Variant analysis parameters L Choose where to run Analvsis configuration Le XE Homo sapiens hg19 sequence 2 Select variant track for Haene eS baki se proband Variant Analysis Genetic Disease Pipeline 3 Variant analysis O Variant Analysis Custom Pipeline specify analysis name Parameters O TEE EE Disease inheritance pattern Other unknown Custom analysis name Naming finpu th date Genetic analysis Press Shift Fi for options 2 Analysis description nput date Genetic analysis Description Press Shift Fi for options Figure 5 3 At this ste
37. ed Note that it is not the minimum value of the probability of the individual variant For the Fixed Ploidy Variant detector if a variant site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold For example if the required CHAPTER 9 WORKFLOWS 36 Identify and Interpret Causal Variants in Family of Four using IVA WES Fixed Ploidy Variant Detection proband i ae targeted region Configurable Parameters le Required variant probability 50 0 Select reads from sibling Ignore broken pairs v Select reads from father Mini coverage Selectreads from mother Minimum count Select reads from proband Minimum frequency Ingenuity Variant gt Locked Settings Analysis for Hereditary Diseases gt Fixed Ploidy Variant Detection proband ses Figure 9 8 Specifying the parameters for the Fixed Ploidy Variant Detection tool variant probability is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair read
38. ics Server CLC Science Server CLC Drug Discovery Server or Bioinformatics Database The plug in also includes Grid Engine Integration __ Proxy Settings Check for Updates Install fromFile Close Figure 2 1 The plugin manager with plugins installed The installed plugins are shown in this dialog To uninstall Click the Ingenuity Variant Analysis Uninstall If you do not wish to completely uninstall the plugin but you don t want it to be used next time you start the Workbench click the Disable button When you close the dialog you will be asked whether you wish to restart the workbench The plugin will not be uninstalled until the workbench is restarted Chapter 3 Introduction to the Ingenuity Variant Analysis plugin The Ingenuity Variant Analysis plugin provides the ability to carry out an Ingenuity Variant Analysis on variant tracks generated in the Workbench and to annotate and filter variants based on information present in the Allele Frequency Community The latest available content from the Ingenuity Knowledge Base and Allele Frequency Community is used in the biological interpretation of input variants from whole genome whole exome targeted amplicon or whole transcriptome sequencing experiments By using published biological knowledge of disease biology the Ingenuity Variant Analysis plugin can be used to prioritize your variants The purpose of the integration is to
39. ing Mutations in Trio WGS fit Identify Variants WGS HD gee Identify and Interpret Causal Variants in Family of Four using IVA WGS Identify and Interpret Causal Variants in Trio using IVA WGS aE fac La Workflows WES 5 E Somatic Cancer WES a a Hereditary Disease WES 2 Filter Causal Variants WES HD F Identify Causal Inherited Variants in Family of Four WES wi identify Causal Inherited Variants in Trio WES fev E wee Identify Rare Disease Causing Mutations in Family of Four WES fev a Identify Rare Disease Causing Mutations in Trio WES E yo Identify Variants WES HD fs Identify and Interpret Causal Variants in Family of Four using IVA WES Identify and Interpret Causal Variants in Trio using IVA WES b Targeted Amplicon Sequencing E A General Workflows TAS EER Somatic Cancer TAS Ee Hereditary Disease TAS my E Filter Causal Variants TAS HD Be Identify Causal Inherited Variants in Family of Four TAS e Identify Causal Inherited Variants in Trio TAS l Ee 3 see Identify Rare Disease Causing Mutations in Family of Four TAS T a Rare Disease Causing Mutations in Trio TAS EF Figure 9 1 Accessing the user settings inside the Ingenuity Variant Analysis web interface The concept of the pre installed ready to use workflows is that read data are used as input in one end of the workflow and in the other end of the workflow you get a track based genome browser view and a table with all
40. ion from 1000 Genomes Project 2 fj Add Information from COSMIC Add Information from ClinVar oe Add Information from Common dbSNP ts Add Information from HapMap gt Add Information from dbSNP Figure 1 The Add Information from Allele Frequency Community tool can be found in the toolbox in the folder Add Information to Variants From Databases If you are connected to a server you will first be asked where you would like to run the analysis If you are not connected to a server the first step is to specify the input for the analysis The Add Information from Allele Frequency Community tool accepts a single variant track FF as input Select the desired variant track as shown in figure 7 2 23 CHAPTER 7 ADD INFORMATION FROM ALLELE FREQUENCY COMMUNITY 24 Select variant track 1 Choose where to se where to run Navigation Area Selected elements 1 2 Select variant track Ela CLC Data GHP Data Se ac oem Variants bbb Sample Variants bbb Sample Variants H A CLC_References Q lt enter search term gt C Batch Figure 7 2 The first wizard step in the Add Information from Allele Frequency Community Select the variant track that you would like to analyze and click on the button labeled Next to go to the next wizard step Click on the button labeled Next to go to the next wizard step where you can specify your Ingenuity username password and the reference sequence as de
41. is many reads are called Minimum count Only variants that are present in at least this many reads are called Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called T Specify the parameters for the Fixed Ploidy Variant Detection tool for the mother and the proband 8 On the last wizard window pressing the button Preview All Parameters allows you to preview all parameters At this step you can only view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Six types of output are generated 3 Reads Track one for each family member 3 Coverage Report Target Region Coverage Report one for each family member 3 Per region Statistics Track Target Region Coverage one for each family member 3 Filtered Variant Track one for each family member An Imported track A URL file Chapter 10 Changing Allele Frequency Community opt in settings In order to gain access to Community Frequency annotations from the Allele Frequency Community your Ingenuity user account must be opted in to the Allele Frequency Community To change your Allele Frequency Community opt in settings carry out the following steps 1 Log in to the Ingenuity Variant Analysis web interface go to http www ingenuity com products variant analysis 2 After logging in go to Settings figure 10 1
42. ity is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 CHAPTER 9 WORKFLOWS Identify and Interpret Causal Variants in Trio using IVA TAS Fixed Ploidy Variant Detection father Select reads from father Configurable Parameters Select reads from mother Required variant probability 50 0 Select targeted region Ignore broken pairs 7 file Minimum coverage Select reads from proband Minimum count Ingenuity Variant Minimum frequency Analysis for Hereditary Diseases QC for Target Sequencing father Fixed Ploidy Variant Detection father mmj Figure 9 23 Specifying the parameters for the Fixed Ploidy Variant Detection tool 48 e Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair reads are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters Minimum coverage Only variants in regions covered by at least th
43. lity of that particular variant might be less than the threshold For example if the required variant probability is set to 0 9 then the individual probability of the variant called might be less than 0 9 as long as the probability of the entire variant site is greater than 0 9 e Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair reads are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters e Minimum coverage Only variants in regions covered by at least this many reads are called e Minimum count Only variants that are present in at least this many reads are called e Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called 4 Specify your reference for the Ingenuity Variant Analysis for Hereditary Diseases as well as your login information figure 9 15 Identify and Interpret Causal Variants in Trio using IVA WGS Ingenuity Variant Analysis for Hereditary Diseases 1 Select reads from father Configur
44. ly view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Six types of output are generated e 3 Reads Track one for each family member 3 Coverage Report Target Region Coverage Report one for each family member 3 Per region Statistics Track Target Region Coverage one for each family member e 3 Filtered Variant Track one for each family member e An Imported track A URL file 9 6 Identify and Interpret Causal Variants in a Trio TAS To run this workflow go to Toolbox Ready to Use Workflows Whole Exome Sequencing f or Targeted Amplicon Sequencing H Hereditary Disease 3 Identify and Annotate Variants in a Trio using IVA TAS 2 CHAPTER 9 WORKFLOWS 46 1 Double click on the Identify and Annotate Variants in a Trio using IVA TAS tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Select the sequencing reads for the father and the mother figure 9 20 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member r Bx Identify and Interpret Causal Variants in Trio using IVA TAS Select sequencing reads 1 Select reads from father apt Navigation Area 2 Select reads f
45. me Browser View Navigation A 2 243 199 373bp v 133 033 480 133 033 500 133 033 520 133 033 540 133 033 56 l 133 033 518 l Range 133 033 471 133 033 562 Sample Variants 2015 08 03 Analysis S IVA Sample 1 Variants 2015 08 03 Analysi Variants 1 308 ple1 v ysi Find E a ASG a E c Track layout Variants track Data aggregation above 10bp wv Reference alleles Dimmed wv lt gt Annotation color i S Hagy ye S Bel n 5 FES Sample Vari X gt Table Settings 4 Ti iew i Rows 12 843 able view Homo sapiens Js ee A Sample coverages Sample zygosities Sample names Conservatio SIFT Function Prediction PolyPhen 2 Community 1000 Geno NHLBI ESP v Sample coverages 12 10 Homozygous Homozygous Sample 1 Variants Sample2 Variants A lt J Sample zygosities 10 10 Homozygous Homozygous Sample 1 Variants Sample2 Variants 18 10 13 86 am lt Sample names 10 10 Homozygous Homozygous Sample 1 Variants Sample2 Variants 14 07 4 Conservation phyloP p value 10 Homozygous Sample 1 Variants 17 11 J SIFT Function Prediction 10 Homozygous Sample 1 Variants 11 00 10 Heterozygous Sample 1 Variants V PolyPhen 2 Function Prediction 10 Heterozygous Sample 1 Variants 14 95 F Community Frequency 10 Heterozygous Sample 1 Variants 10 Heterozygous Sample 1 Variants v 1000 Genomes
46. mp The analysis name is the name that is shown on the Ingenuity Variant Analysis page as the name when you choose My Analyses The same analysis name can furthermore be used in a Variant Analysis Custom Pipeline if you specify it in the Custom analysis name field See above e Analysis description This will be the description of the analysis in Ingenuity Variant Analysis once created There are a few shorthand notations available input will be substituted with the name of the input experiment date is substituted with a date stamp Click on the button labeled Next to go to the next wizard step where you must specify family data for the analysis figure 5 4 e Gender of proband Select the gender of the individual affected by the disease e Variant track for father mother Select the variant track for the father or mother as appropriate The variant track for at least one parent must be specified e Father mother affected Once you have selected a variant track for a parent the option to set the disease status of that parent will be enabled Check this box if the given parent is affected by the same disease as the proband Uncheck this box if the given parent is not affected Ingenuity Variant Analysis for Hereditary Diseases Family information 1 Choose where to run Proband Select variant track for ana Gender of proband Female 3 Variant analysis Father Parameters Variant track for father FF Sample Variants
47. ncing reads 1 Select reads from fath a Navigation Area Selected elements 1 Sj CLC_Data a Father workflows t3 Trio i Affected child zF ather Mother Q7 lt enter search term gt Figure 9 13 Specify the sequencing reads for each family member successively 3 Specify the parameters for the Fixed Ploidy Variant Detection tool for the proband figure 9 14 Identify and Interpret Causal Variants in Trio using IVA WGS Fixed Ploidy Variant Detection proband Configurable Parameters 2 Select reads from mother Required variant probability 50 0 1 Select reads from father 3 Select reads from proband Ignore broken pairs 7j Minimum coverage 10 4 Fixed Ploidy Variant Detection proband Minimum count Minimum frequency gt Locked Settings Figure 9 14 Specifying the parameters for the Fixed Ploidy Variant Detection tool The parameters that can be set are e Required variant probability is the minimum probability value of the variant site required for the variant to be called Note that it is not the minimum value of the CHAPTER 9 WORKFLOWS 41 probability of the individual variant For the Fixed Ploidy Variant detector if a variant site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probabi
48. ngenuity VA username email address xxxx xxxx com Ingenuity VA password PTET iti iii 3 Family information 2 aa i Accountinformaton Previous gt Next Figure 5 6 Specify the account information your Ingenuity username email address and password are required at this step username or password you must first create an Ingenuity account Account creation is described in chapter 3 Click on the button labeled Next to go to the last wizard step figure 5 7 where you can set the output options If the Import annotated and filtered variants option is checked the tool will produce a variant track as output If it is unchecked the analysis will be created and can be accessed inside the Ingenuity Variant Analysis web interface but the results will not be imported into the workbench Note it is not possible to import results if you have selected the Upload only pipeline earlier in the wizard lf you choose to open the results the two generated outputs will be opened in the View Area without being saved In this case you will have to manually save the outputs if you would like to keep them If you choose to save the outputs click on the button labeled Next to specify where to save the results and click on the button labeled Finish to start the Ingenuity Variant Analysis Your results will not be opened automatically but will be saved at the destination you have specified CHAPTER 5 INGENUITY VARIANT ANALYSIS FOR HEREDITAR
49. nterpret Causal Variants in a Trio using IVA TAS Furthermore the plugin includes the possibility to update a variant track containing the results of an Ingenuity Variant Analysis if you change the filtering settings inside the Ingenuity Variant Analysis web interface Access to Ingenuity Variant Analysis requires a Subscription However a trial period is available that allows the analysis of up to 4 samples The first step is to register for an Ingenuity Variant Anal ysis account https apps ingenuity com isa account signup va utm_source ingenuity utm_medium banner utm_campaign webpage preview Upon completion of registration you will receive an email to activate your new Ingenuity Variant Analysis account Once you ve logged in for the first time and accepted the End User License Agreement you can use these credentials to allow the plugin to send variant data from Biomedical Genomics Workbench to Ingenuity Variant Analysis If you opt into the Allele Frequency Community you will get a month of free analysis without a subscription to Ingenuity Variant Analysis See chapter 10 to change your Allele Frequency Community opt in status Chapter 4 Ingenuity Variant Analysis The Ingenuity Variant Analysis tool is installed in the toolbox in a folder called Ingenuity Variant Analysis as shown in figure 4 1 The Ingenuity Variant Analysis tool L can be launched from the toolbox from the folder Ingenuity Variant Analysis Ingen
50. ons Cancer Useful if you re seeking to identify cancer driver variants If this option is selected the type of cancer must be specified in the Disease name drop down menu Stratification study Useful if you have two groups of samples and are looking for variants that distinguish the two Variant Analysis Custom Pipeline Useful if you have already carried out an Ingenuity Variant Analysis where you have set up a desired filtering cascade and want to re use the same filtering cascade for a new analysis If this option is selected the name of the custom analysis must be specified in the Custom analysis name field The name you enter in the Custom analysis name field must match the Name field of an existing analysis in Ingenuity Variant Analysis exactly as it appears in the Ingenuity Variant Analysis web interface Note it is recommended that you provide unique names to all your analyses in Ingenuity Variant Analysis Upload only Useful if you just want to upload samples and do not wish to carry out an analysis Note in this case no results will be downloaded CHAPTER 4 INGENUITY VARIANT ANALYSIS 11 e Analysis name The name of the analysis You can enter a name of your own choice by typing in the name or by using the options that appear when you press Shift F1 The available shorthand notations are input will be substituted with the name of the input experiment date is substituted with a date stamp The
51. p you can specify the analysis parameters for the Ingenuity Variant Analysis for Hereditary Diseases tool Variant Analysis Custom Pipeline useful if you have already carried out an Ingenuity Variant Analysis where you had set up a desired filtering cascade and want to re use the same filtering cascade for a new analysis If this option is selected the name of the custom analysis must be specified in the Custom analysis name field The name you enter in the Custom analysis name field must match the Name field of an existing analysis in Ingenuity Variant Analysis exactly as it appears in the Ingenuity Variant Analysis web interface Note it is recommended that you provide unique names to all your analyses in Ingenuity Variant Analysis Upload only useful if you just want to upload samples and do not wish to carry out an analysis Note in this case no results will be downloaded e Disease inheritance pattern The disease inheritance pattern applicable to the disease you are studying Supported modes are Dominant Recessive X linked De novo or Other Unknown CHAPTER 5 INGENUITY VARIANT ANALYSIS FOR HEREDITARY DISEASES 15 e Analysis name The name of the analysis You can enter a name of your own choice by typing in the name or by using the options that appear when you press Shift F1 The available shorthand notations are input will be substituted with the name of the input experiment date is substituted with a date sta
52. pends on the technology you used for sequencing Identify and Interpret Causal Variants in Trio using IVA WES Select input for targeted region file Navigation Area Selected elements 1 targeted_sequencing a E 50293689_Regions_BED CTFR Cergentis AmpliSeq agilent_sure_select 1 Select targeted region file 5 4450293689_Regions_BED Ww j Q lt enter search term gt Figure 9 17 Specify a target region file 4 Select the sequencing reads for the proband 5 Specify your reference for the Ingenuity Variant Analysis for Hereditary Diseases as well as your login information figure 9 18 The parameters that can be set are e Reference Select the genome sequence you would like to work with usually hg19 e Analysis pipeline name specify which kind of analysis you would like to perform on your variants Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a customized pipeline available in your Ingenuity IVA account Upload only does not carry out an analysis just upload samples to the Ingenuity Variant Analysis Choose this option if you wish to run the analysis or create a customized pipeline on the Ingenuity web interface CHAPTER 9 WORKFLOWS 44 Identify and Interpret Causal
53. quency Community This can happen when the variant is a new one in the Allele Frequency Community database but also for reference variant from an heterozygous pair whose non reference variant was kept by the Remove Variants found in Allele Frequency Communitytool as no reference variants are ever found in the Allele Frequency CHAPTER 8 REMOVE VARIANTS FOUND IN ALLELE FREQUENCY COMMUNITY 29 Community database To filter out reference variants click on the Filter button in the variant table select Reference allele in the drop down menu and keep only the variants that contain No After this step the only variants without an annotation in the Community frequency column will be the ones considered as new variants i e not previously found in the Allele Frequency Community database Chapter 9 Workflows Installing the Ingenuity Variant Analysis plugin will also add six ready to use workflows to the Ready to Use Workflows section of the toolbox under the Whole Genome Sequencing Whole Exome Sequencing and Targeted Amplicon Sequencing folders figure 9 1 poe Whole Genome Sequencing ng E General Workflows WGS E Fa A Somatic Cancer WIGS f Hereditary Disease WGS iG Filter Causal Variants WGS HD fee a Identify Causal Inherited Variants in Family of Four WGS Identify Causal Inherited Variants in Trio WGS been ES wee Identify Rare Disease Causing Mutations in Family of Four WGS yes Identify Rare Disease Caus
54. r Hereditary Diseases The Ingenuity Variant Analysis for Hereditary Diseases tool is installed in the toolbox in a folder called Ingenuity Variant Analysis as shown in figure 5 1 The Ingenuity Variant Analysis for Hereditary Diseases tool T can be launched from the toolbox from the folder Ingenuity Variant Analysis f 3 Ingenuity Variant Analysis A Ingenuity Variant Analysis g Ingenuity Variant Analysis for Hereditary Diseases Figure 5 1 The Ingenuity Variant Analysis for Hereditary Diseases tool can be found in the toolbox in a folder called Ingenuity Variant Analysis If you are connected to a server you will first be asked about where you would like to run the analysis If you are not connected to a server the first step is to specify the input for the analysis The Ingenuity Variant Analysis for Hereditary Diseases tool accepts a single variant track Ft as input Select the desired variant track as shown in figure 5 2 The variant track selected in this step will be considered the proband i e the individual affected by the disease you are studying Click on the button labeled Next to go to the next wizard step where you can set the analysis parameters as described below figure 5 3 e Reference Select the human reference sequence that is found under CLC_References in the Navigation Area Only complete human genomes can be used as references We currently support the human reference genome hg19 The use of sel
55. ressing the button Preview All Parameters allows you to preview all parameters At this step you can only view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Six types of output are generated e 4 Reads Track one for each family member e 4 Coverage Report Target Region Coverage Report one for each family member e 4 Per region Statistics Track Target Region Coverage one for each family member e 4 Filtered Variant Track one for each family member CHAPTER 9 WORKFLOWS 40 e An Imported track e A URL file 9 4 Identify and Interpret Causal Variants in a Trio WGS To run this workflow go to Toolbox Ready to Use Workflows Whole Genome Sequencing Hereditary Disease ap Identify and Annotate Variants in a Trio using IVA WGS Webs 1 Double click on the Identify and Annotate Variants in a Trio using IVA WGS tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Select the sequencing reads for the father mother and proband respectively figure 9 13 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member Identify and Interpret Causal Variants in Trio using IVA WGS Select seque
56. rom mother J A CLC_Data workflows ta Trio 4 Affected child i Father Qy lt enter search term gt Previous gt Next Figure 9 20 Specify the sequencing reads for each family member successively 3 Specify a target region file figure 9 21 This is a file that depends on the technology you used for sequencing Identify and Interpret Causal Variants in Trio using IVA TAS Select input for targeted region file Navigation Area Selected elements 1 B targeted_sequencing F E 50293689_Regions_BED CTFR Cergentis AmpliSeq agilent_sure_select 5 4450293689_Regions_BED mm 1 Select targeted region file K r Q lt enter search term gt ECI Figure 9 21 Specify a target region file 4 Select the sequencing reads for the proband 5 Specify your reference for the Ingenuity Variant Analysis for Hereditary Diseases as well as your login information figure 9 22 The parameters that can be set are e Reference Select the genome sequence you would like to work with usually hg19 e Analysis pipeline name specify which kind of analysis you would like to perform on your variants Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants Variant Analysis Custom Pipeline specify analysis name chose this option if you want to run a
57. s are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters Minimum coverage Only variants in regions covered by at least this many reads are called Minimum count Only variants that are present in at least this many reads are called Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called 6 On the last wizard window pressing the button Preview All Parameters allows you to preview all parameters At this step you can only view the parameters it is not possible to make any changes Choose to save the results and click on the button labeled Finish Six types of output are generated 4 Reads Track one for each family member 4 Coverage Report Target Region Coverage Report one for each family member 4 Per region Statistics Track Target Region Coverage one for each family member 4 Filtered Variant Track one for each family member An Imported track A URL file CHAPTER 9 WORKFLOWS 37 9 3 Identify and Interpret Causal Variants in a Family of Four TAS To run this workflow go to Toolbox Ready to Use Workflows Whole Exome Sequencing f4 or
58. s filter cascade that narrows down the initial number of variants to focus on a limited number of specific variants that are left after applying a number of different filters After running the initial analysis from the Workbench add more filters with the button labeled Add Filter found at the bottom of the filter cascade Modify or delete filters with the paper icon found in the right hand side of the individual filters in the filter cascade figure 6 6 Click on the information icon next to the paper icon to get more information about Ingenuity Variant Analysis CHAPTER 6 ANALYSIS USING THE PLUGIN AND THE IVA WEB INTERFACE 21 x Common Variants a0 5084 1522 t x Predicted Deleterious A 3633 1479 t Figure 6 6 Click on the paper icon red arrow if you would like to delete the filter see the filter details or if you would like to adjust them Click on the information icon blue arrow if you would like to learn more about Ingenuity Variant Analysis When you have modified the filters on the Ingenuity Variant Analysis web page you can either choose to use the options provided on the Ingenuity Variant Analysis web page to go into detail with the individual variants or you can go back to the Workbench and visualize the variants in the Genome Browser View The modified variant track can be imported into the Workbench by right clicking on the original Ingenuity Variant Analysis variant track output that was generated with the default
59. s well as your login information figure 9 4 Identify and Interpret Causal Variants in Family of Four using IVA WGS 28 Ingenuity Variant Analysis for Hereditary Diseases 1 Select reads from sibling Configurable Parameters 2 Select reads from father Reference X Homo sapiens hg 19 sequence 3 Select reads from mother Analysis pipeline name Variant Analysis Genetic Disease Pipeline 4 Select reads from proband Disease inheritance pattern Other unknown Custom analysis name Fixed Ploidy Variant Detection sibling Gender of proband Other Unknown Ingenuity Variant Foe moe Analysis for Hereditary Mother affected Diseases Sibling 1 affected Gender of sibling 1 Other Unknown Analysis name bene ams emi Analysis description input date Description Press Shift F1 tions Ingenuity VA username email address cxxx dxxx giagen com Ingenuity VA password gt Locked Settings j Previous gt Next Finish X Cancel__ Figure 9 4 Specify a reference and login information to Ingenuity Variant Analysis The parameters that can be set are Reference Select the genome sequence you would like to work with usually hg19 Analysis pipeline name specify which kind of analysis you would like to perform on your variants CHAPTER 9 WORKFLOWS 33 Variant Analysis Genetic Disease Pipeline pipeline available on the Ingenuity web interface to identify causal variants
60. scribed below figure 7 3 Reference and Account information 1 Choose where to run 2 Select variant track Account information z PE Account Ingenuity username email address s900 20000 com Ingenuity password Reference information Y Finish Figure 7 3 At this step you can specify the analysis parameters for the Add Information from Allele Frequency Community tool e Ingenuity username email address used to log in to Ingenuity Variant Analysis e Ingenuity password password corresponding to your Ingenuity username e Reference select the human reference sequence that is found under CLC_References in the Navigation Area Only complete human genomes can be used as references We currently Support the human reference genome hg19 The use of selected regions of the genomes e g individual chromosomes is not supported Click on the button labeled Next to go to the final wizard step figure 7 4 where you can set the output options CHAPTER 7 ADD INFORMATION FROM ALLELE FREQUENCY COMMUNITY 25 Add Information from Allele Frequency Community Result handling Choose where to run 2 Select variant track 3 Reference and Account Result handling information Open Result handling Save Log handling Open log Previous Figure 7 4 The result handling step in the Add Information from Allele Frequency Community wizard If you choose to open the results
61. sexual organs that are not yet fully developed and unknown e Check if an other family member is affected the mother the father or the proband e If the sibling is affected specify its gender e Analysis name choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Analysis e Analysis description choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 6 Specify the parameters for the Fixed Ploidy Variant Detection tool for the father mother and proband successively figure 9 23 The parameters that can be set are e Required variant probability is the minimum probability value of the variant site required for the variant to be called Note that it is not the minimum value of the probability of the individual variant For the Fixed Ploidy Variant detector if a variant Site and not the variant itself passes the variant probability threshold then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold For example if the required variant probabil
62. tTutorials CHAPTER 6 ANALYSIS USING THE PLUGIN AND THE IVA WEB INTERFACE 22 i Table Settings v Sample names t Conservation phyloP p value Known variation Variant validated other experiment Select All Od Figure 6 8 You can see which columns have been added in the Table view of the variants Chapter 7 Add Information from Allele Frequency Community The Add Information from Allele Frequency Community tool allows you to add Community Frequency annotations from the Allele Frequency Community to variant tracks To be able to obtain Community Frequency annotations from the Allele Frequency Community your Ingenuity user account must be opted in to the Allele Frequency Community Chapter 10 describes how to change your Allele Frequency Community opt in status The Add Information from Allele Frequency Community tool is installed in the toolbox in the following location figure 7 1 Add Information to Variants From Databases Add Information from Allele Frequency Community 4 Toolbox 4 Add Information to Variants oe Add Information from Variant Databases AEE Add Conservation Scores Le Add Exon Number LTT Add Flanking Sequence RR Add Fold Changes T Add Information about Amino Acid Changes ee Add Information from Genomic Regions 4 Add Information from Overlapping Genes A Link Variants to 3D Protein Structure fie Download 3D Protein Structure Database f From Databases ve Te Add Informat
63. tcut and choose Run as Administrator Then follow the procedure described below 4 CHAPTER 1 INSTALLATION OF THE INGENUITY VARIANT ANALYSIS PLUGIN Manage Plugins and Resources Additional CLC bio support cicbio com Version 1 5 1 Build 131211 2142 102901 Perform alignments with ClustalO ClustalW and MUSCLE ee Download and Install Annotate with GFF file Q CLC bio support ckbio com Version 2 2 6 Build 131211 2143 102901 Using this plug in it is possible to annotate a sequence from list of annotations found in a GFF file Located in the Toolbox Batch Rename Q CLC bio support ckbio com Version 1 3 1 Build 131211 2144 102901 Rename files in batch by adding a prefix or a number Biobase Genome Trax Annotate Q CLC bio su io com Version 2 0 11 Build 140103 1321 103719 Create tracks with various data from Biobase Genome Trax Q CLC bio support ckcbio com Version 2 0 11 Build 140103 1322 103719 Create tracks with various data from Biobase Genome Trax Plugin requires registration Blast2GO PRO Q BioBam Bioinformatics pluginsupport blast2go com CD additional Alignments This module allows for use of two other alignment methods which are otherwise not distributed with the CLC Workbench When the plug in is installed you will see the new alignment methods in the Toolbox under Alignments and Trees gt Additional Alignments When you run the alignments there are a number of parameters that can b
64. the identified variants subjected to the Ingenuity Variant Analysis These six workflows seek to identify and interpret causal variants in either a family of three the 30 CHAPTER 9 WORKFLOWS 31 proband and his two parents or a family of four the proband his parents and a sibling Once you have selected the workflow in the folder relevant to your input data you can read the steps you need to take to start the workflow For more information on the specific tools used in this workflow see the Biomedical Genomics Workbench manual chapter on Workflows 9 1 Identify and Interpret Causal Variants in a Family of Four WGS To run this workflow go to Toolbox Ready to Use Workflows Whole Genome Sequencing Hereditary Disease a Identify and Annotate Variants in a Family of Four using IVA WGS PE 1 Double click on the Identify and Annotate Variants in a Family of Four using IVA WGS tool to start the analysis If you are connected to a server you will first be asked where you would like to run the analysis 2 Select the sequencing reads for the sibling father mother and proband successively figure 9 2 You can do that by double clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard Click on the button labeled Next between each family member Identify and Interpret Causal Variants in Family of Four using IVA WGS
65. ther Required variant probability 50 0 3 Select targeted region Ignore broken pairs v file Minimum coverage Select reads from proband Minimum count at uity Variant Minimum frequency Analysis for Hereditary Diseases gt Locked Settings Fixed Ploidy Variant Detection father a Previous gt Next Figure 9 19 Specifying the parameters for the Fixed Ploidy Variant Detection tool e Ignore broken pairs When ticked reads from broken pairs are ignored Broken pairs may arise for a number of reasons one being erroneous mapping of the reads In general variants based on broken pair reads are likely to be less reliable so ignoring them may reduce the number of spurious variants called However broken pairs may also arise for biological reasons e g due to structural variants and if they are ignored some true variants may go undetected Please note that ignored broken pair reads will not be considered for any non specific match filters e Minimum coverage Only variants in regions covered by at least this many reads are called e Minimum count Only variants that are present in at least this many reads are called e Minimum frequency Only variants that are present at least at the specified frequency calculated as count coverage are called On the last wizard window pressing the button Preview All Parameters allows you to preview all parameters At this step you can on
66. uity Variant Analysis in Ingenuity Variant Analysis i im Ingenuity Variant Analysis for Hereditary Diseases Figure 4 1 The Ingenuity Variant Analysis tool can be found in the toolbox in a folder called Ingenuity Variant Analysis If you are connected to a server you will first be asked about where you would like to run the analysis If you are not connected to a server the first step is to specify the input for the analysis The Ingenuity Variant Analysis tool accepts variant tracks FF as input Select the desired variant track or several variant tracks as input as shown in figure 4 2 All the variant tracks selected in this step are assumed to be case samples Bx Ingenuity Variant Analysis Select variant tracks 1 Choose where to run Navigation Area Selected elements 1 2 Select variant tracks g Sat ac Data HE Sample1 Variants Hk Sample2 Variants ib Sample3 Variants El EA CLC_References m i Qr lt enter search term gt Batch gt Next Finish Cancel Figure 4 2 The first wizard step in the Ingenuity Variant Analysis Select the variant track that you would like to analyze and click on the button labeled Next to go to the next wizard step Click on the button labeled Next to go to the next wizard step where you can set the analysis 9 CHAPTER 4 INGENUITY VARIANT ANALYSIS 10 parameters as described below figure 4 3 Ingenuity Variant
67. wizard followed by the date and the word Analysis e Analysis description choose a name for your analysis The default name is the name of the first input file selected in the wizard followed by the date and the word Description e Ingenuity VA username usually the email address you used to sign in the Ingenuity Variant Analysis e Ingenuity VA password the password you chose when you signed in on the Ingenuity website 5 Specify the parameters for the Fixed Ploidy Variant Detection tool for the proband mother sibling and father successively figure 9 12 The parameters that can be set are CHAPTER 9 WORKFLOWS 39 Identify and Interpret Causal Variants in Family of Four using IVA TAS Fixed Ploidy Variant Detection proband Configurable Parameters Select targeted region file Required variant probability 50 0 Select reads from sibling Ignore broken pairs v Select reads from father Mini coverage 10 Select reads from mother Minimum count 5 Select reads from proband Minimum frequency Ingenuity Variant gt Locked Settings Analysis for Hereditary Diseases Fixed Ploidy Variant Detection proband ess Figure 9 12 Specifying the parameters for the Fixed Ploidy Variant Detection tool e Required variant probability is the minimum probability value of the variant site required for the variant to be called Note that it is not the minimum value of the
68. wser View in the Workbench The identified variants can be viewed in track format in the Genome Browser View by double clicking on the name of the variant track in the Navigation Area The button labeled Create Genome Browser View in the upper right corner of the View Area can be used to create a list of tracks in the same view which allows comparison of the identified variants with other tracks such as the reference sequence the CDS read mappings or other variant tracks e View the variants on the Ingenuity Variant Analysis web page This option allows adjustment of the predefined filter settings The variants in Ingenuity Variant Analysis can be accessed in two different ways 1 Use the link provided in one of the output files in the Workbench Copy the link and paste it into an internet browser This will Send you directly to the variant analysis on the Ingenuity Variant Analysis web page An example is shown in figure 6 3 My Samples My Analyses Publications IVA analysis of candidate variants and genes x OJ Feedback Summary Variants Genes Groups Complexes Pathways Processes Diseases Overview Stare EE A Edit Columns Create List 2863 variants x Confidence F Chr Position Gene Region Gene Symbol Protein Variant Case Samples Translation Impact SIFT Functio Regulatory Site Regulator Variant Findings dbSNP ID 6107 1524 1 6324710 Exonic ACOT7 p R313W p R32 m missense Damaging 4 A

Download Pdf Manuals

Related Search

Related Contents

    Kaspersky Lab Small Office Security 2.0, 1s, 5u, 1y, Base, Box, ENG  バッテリーウェルダー  Quick start guide - Diamond Multimedia  Philips CTX525SLV 3" 108g Silver  Samsung 22" бизнес монитор със страхотна ергономична функция Наръчник за потребителя  

Copyright © All rights reserved. Failed to retrieve file