User Manual
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1. Samtools DNANexus 95 59 95 28 94 80 95 03 Precision Rate 82 18 85 37 88 45 84 31 Sensitivity 98 96 98 85 98 68 98 80 0 000 20 00 40 00 60 00 80 00 98 96 Precision Rate TP TP FP Sensitivity TP TP FN Specificity TN TN FP Specificity As one may see on Fig 28 the iBinom solution demonstrates results comparable to the widely applied solutions and even outperforms them in Precision rate and Specificity Annotation Annotation is performed in two key steps First we annotate the variants followed by prediction of their coding effects on known genes such as synonymous or non synonymous SNPs start codon gains or losses stop codon gains or losses Also for each variant we evaluate its genomic locations within intronic 5 UTR 3 UTR upstream downstream or intergenic regions Second the iBinom machine learning algorithm is used to define the level of variants pathogenicity called iBinom score The iBinom score varies from 0 to absolutely non pathogenic mutation to 1 for extremely pathogenic mutation All variants with the iBinom score exceeding 0 9 should be considered as potentially clinically significant For the purpose of algorithm training procedure we used information from the Page 23 iBinom Quick Start Guide databases and predictors listed below The most generally recognized and reputed scores values and databases information are represented on the Filteri2. To see the variant and gene details click on the variant All available information will be shown in the pop up window as on Fig 22 lt Patients Report e Filter Click on to filter AMPLE Heter region p Gin4 Caa T FCGR Heter coding p Leul cTc c Variant details Gene info Chr Position Ref Alt Zygosity Gene Rsid Transcript id Amino acid change Quality Depth Effect Variant type Quality filter 115236057 G het AMPDI rs17602729 ENST000005201 13 p Gin45 c 133C gt T 175 009 21 STOP_GAINED SPLICE_SITE_REGION SNP EndDistBias Filter saving and variant report creating yakovishinad gmail com Frequency kk afrk amrk eask eurk sasae aae eaxx afrx _amrx_adjx_eas x_finx_nfex_othx_sast ENST000005201 13 rs 7602729 pos 1 115236057 Frequency kk_afrk_amrk_eask_eurk_sase_aae eaxx_afrx_ amr x_adjx_eas x_finx_nfex_othx_sas ENST00000367969 rs10127939 pos 1 161518333 To remember your filtration parameters click on the grey button Save filter fill the filter name and description optional fields in the pop up window and press Save as shown on Fig 23 Thus you will be able to apply at one click the same filtration parameters for your consecutive analyses Patients Report Filter Click on to filter Save filter Close f kovishinad gmail com Q Q To load the filter simply click Load filter and choose your
3. applies to ensure the variant quality ex read depth gt 10 p value of biases gt 0 05 lt Patients Report GO yakovishinad gmail com Filter Depth and quality Add to filter X Depth and quality Depth Diseases and genes Quality Chromosome position rsID Quality filter passed Variant type zygosity effect Scores 2 Selecting diseases and gene panels creating a custom panel Panel is the list of genes that have been associated with a given disease Genomic panels were identified based on information in the Online Mendelian Inheritance in Man OMIM catalog2 To select the panels from the list tick the boxes next the panels of interest To filter by genes of your interest type a list of genes in the field Gene You may create your own panel as explained on Fig 11 lt Patients Report yakovishinad gmail com Depth and quality Filter and genes Add to filter X Gene Diseases and genes Genes from panels Chromosome position rsID Variant type zygosity effect Custom panels yx Custom panel name Scores ACMG Incidental Findings j Gene symbols demie bts om Cardiological Disease y Clinvar Ear Nose and Throat Diseases Add custom panel Liver Kidney and Endocrinological Diseases To add a custom panel please name it and list genes separated with commas in the related fields then click the button Add custom panel Your custom panel will appear on the screen as displayed on Fig 1
4. been completed you may access the results and create a medical report By default a report will have the same name as the corresponding input file You may delete the file or rename it by mousing over the patient and clicking cross or pencil button that will appear respectively as displayed on Fig 7 Page 8 iBinom Quick Start Guide 17 june 2015 x f John Johnson VCF CSV Quality control To download VCF CSV or Quality Control report corresponding to the sample navigate and select the required option To view and start variant filtering simply click on the sample name you will be redirected to the Filtering page At the start all the variants contained in VCF file are displayed on the Filtering page To filter the candidate variants from all the rest you may take advantage of filtering features On the Filtering page you will find a left bar with the filtering options along with the interactive filtering window By clicking on at the blue box as shown on Fig 8 the left filtering bar is activated and you are able to select the options and sort variants of your interest E Patients Report O ignat 990 gmail com Filter Click on to filter Q 3 44553 variants m 2 Download CSV Create report SAMDI I SNP Homozygote intron Scores Frequency AG kk_afrk amrk _eask eurk sasxx afrx amrx adjx easx f inx_nfex _othx sas Depth ACMG ENST00000342066 II benign rs4372192 pos 1 876499 c 707 25A gt G iBinom Quick Start
5. filter as on Fig 24 Patients Report Filter Click on to filter Deptt nda f Diseases and gene Inp iBinom Quick Start Guide Load filter Your current filtration will be lost due to loading saved filter Filter Test Filter Load filter I Load BN A Page 18 In order to download the most relevant variants remained after applied filtration steps the system enables you to generate the iBinom variant report Click on Create report button as shown on Fig 25 and the system will automatically save and open the PDF report file lt Patients Report O av ibinom com 3 Filter Click on to filter Populations Edit gt E 3 variants sample Create report FMO3 SNP ere Heterozygote non synonymous coding Scores Frequency MT psC7vr CDDp F MMr PP3V MAr plP7vr SFTr kk_afrk_amrk_eask eurk sasae aae eaxx afrx amrx a djx_easx_finx_nfex_othx_sast p Glul58Lys c 472G gt A Clinvar ENST00000367755 rs2266782 Gag Aag Pathogenic pos 1 171076966 Heterozygote non synonymous coding Scores Frequency MT psC7vr CDDp F MMr PP3V MAr plP7vr SFTr kk_afrk amrk_eask eurk sasae aae eaxx_afrx_amrx_a Personal account settings In order to access your personal account settings click to your account e mail shown in the top right corner of the screen Type in a new e mail or password if you wish to modify them and click on the button Save to apply the changes Fig 26 lt Patenrs Settings a Tell us
6. start using the service you should create your personal account Please proceed with the following instructions Visit iBinom homepage at https www ibinom com 1 Click the blue button Try now You will see a window with a registration form 2 Fill registration fields with your personal details Communicate to us how you got aware about our service If you are a Clinical Genomics Training Center s trainee tick a box next to the CGTC name 3 Create your password you will have to enter it each time you access the service Once the registration form is completed you will receive an account activation email 4 Follow the link provided in the e mail to complete your registration You will be redirected to the iBinom website and logged in automatically Congratulations Your personal iBinom account has been created All the data submitted to your account including your personal details are secured and available only to the account holder Please do not communicate your password to anyone else in order to keep your privacy Once registered you will be able to perform 6 panel or 6 exome VCF file analyses and 2 panel or 2 exome FASTQ BAM file analyses free of charge Logging in to your account You can always access your iBinom account from the homepage at https www ibinom com by clicking the Login button After entering your login and password you will be directed to the Dashboard page of your account If y
7. 2 Page 11 iBinom Quick Start Guide E Patients Report yakovishinad gmail com Diseases and genes Add to fiter X Depth and quality Gene Diseases and genes Genes from panels Chromosome position rsID Variant type zygosity effect Custom panels Custom panel name Scores ACMG Incidental Findings Gene symbols Cardiological Disease Clinvar Ear Nose and Throat Diseases Add custom panel Liver Kidney and Endocrinological Diseases Populations frequency You may create as many custom panels as you wish They all will be displayed on the screen once you go for Custom panels To delete a panel click on a cross on the right side next to the panel name In order to proceed with filtering tick the box next to a created panel and press Add to filter 3 Selecting chromosomes positions rsID The Tab Chromosomes positions rsID enables choosing chromosomes position or rsiDs Rapid Stain Identification Series of interest Chromosomes can be chosen either separately or by groups Autosomal 1 22 group and Sex X and Y group In order to choose a certain chromosome position at first choose a chromosome and enter a position in the following format 1000 2000 To specify an rsID just enter list of comma separated rsiDs to the corresponding field as shown on Fig 13 lt Patients Report O yakovishinad gmail com Filter Chromosome position rsiD Add to filter X Depth and quality Chromosome Autos
8. Guide Alignment benchmarking We have benchmarked the most applied open source read mappers The study employed the public server for genome comparison and analytic testing GCAT http www bioplanet com gcat Benchmarking has been performed on four single end testing datasets offered by GCAT server BWA MEM tool proved to be the most efficient and accurate mapper Results are represented in the table below Name Total Correct Incorrectly Mapped Unmapped Reads Reads Reads BWA MEM 7863529 101527 pene fase es Variant calling Before starting variant calling alignment filtering is applied First non mapped reads and reads with low mapping quality are filtered out Then all remaining reads are grouped Two reads are connected in one group if they intersect and this connection is transitive between the reads When all reads are grouped the obtained groups are sorted out by the maximum coverage depth in a group by amount of reads and amount of covered nucleotides within a group The step helps to remove low covered regions preventing from false positive variants Also this step filters out clusters that are not located within the regions of interest for targeted or exome sequencing Variant calling is performed using SAMtools libraries with tuned settings and adjusted to be run on MapReduce cluster SAMtools algorithm uses Bayesian Page 21 Binom Quick Start Guide model to detect variants and its quality values Local
9. Guide Filtering options Once you have accessed the Filtering page you may sort variants by the following options e Depth and quality e Diseases and genes e Chromosome position rsiD e Variant type zygosity effect e Scores e Population frequencies e Databases lt Patients Report O ignat 990 gmail com fer Depth and quality Depth For each option you may select certain parameters Please remember to click on the Add to filter button to apply the selected parameters to the filter as shown on Fig 9 otherwise the system will not proceed with it 1 Selecting depth of coverage and mapping quality Depth of coverage indicates how many times a given base was sequenced i e how many reads cover the base If a coverage value is low in comparison with one allele coverage this SNP might be generated because of sequencing or mapping errors and should be treated with caution Mapping quality indicates the minimum mapping quality of the variants that is accepted to the filtering process The parameter shows how probable is the variant position 20 means that read is mapped correctly with a probability of 99 To change depth or quality parameter mouse on gt 20 and type in a new value see Fig 10 If you unsure about the appropriate parameters tick the box next to Quality filter passed Mouse on the field name to see the exact preconfigured Page 10 Binom Quick Start Guide values the system
10. O BINoMm iBinom Quick Start Guide Contents NCAA a a o te Peer ern eens 3 Sineme UP or IBIROM ACCOUNT AR NA RU Me attentes etes e ot tele 4 LOR FINE INTO YOUN ACCOUNT na anna E a 4 Getme started ai ashes circ A AS 5 SHS IB OM din 7 Unidades VOU atari pots 7 Startine data ana A A A A ia 8 Accessing th resultsand Miterine Pati a 8 F WESTIN ODON id aa cod 10 1 Selecting depth of coverage and mapping quality ss 10 2 Selecting diseases and gene panels creating a custom panel 11 3 Selecting chromosomes positions FSID cccccecccsscceseceseeceeceeeeceseceeueeaecseeeeeuceeeceseeeeaeeeeeeseeeseneeaaeees 12 4 Selecting variant type zygosity and effect sise seessssenssseessennns 12 Dis DEIOCEINE SCORES SE Ne O 13 6 SEIECLING POD UlatION TREG UCI CY ves RSR A ef ut 14 TSE letting CIN Val parameter ias 15 General Ver USC AS oros 16 Filter savine and varant report creatine dd e e dd do 18 Personal ACC OUI se tner dad did 19 BI oman alys Stechanical detalles 20 Generar desen RSR A ES 20 PCD PO Cessna adds 20 Fraements alsnment sarria 20 Allenment penca MARINE A AS AER 21 Varant calin cassone Rd ee ee ieadae 21 Marant caline D nCchMarRiINB added 22 PINOT HO Masacran 23 Page 2 Binom Quick Start Guide Introduction Welcome to the iBinom genome data analysis service Aiming to get a foothold in the emerging clinical next generation sequencing NGS market iBinom launched a simple fast and affordabl
11. about a problem via email av ibinom com ogout Setti ngs Personalize your iBinom account Email and notifications Password Sharing raw data with colleague Email Save Password Save Page 19 iBinom Quick Start Guide iBinom analysis technical details General design Sequencing data analysis consists of 5 steps Preprocessing Reads alignment Variants calling Variants annotation ee A S a Report creating Data uploaded to the server are protected with HTTP Secure https protocol We use the human genome reference version GRch37 75 hg19 on each step of data analysis Preprocessing Before the alignment process we calculate fragments quality distribution Low quality reads are filtered out from later processing The following statistics are calculated on filtered reads e Distribution of nucleotides phred qualities e Average quality per position within read e Distribution of reads lengths e Amount of each nucleotide in data All statistics are provided in the iBinom Quality Report Fragments alignment To align the reads iBinom uses BWA MEM tool adjusted to run on MapReduce cluster of Elastic MapReduce Amazon system The BWA MEM alignment algorithm is based on Burrows Wheeler transformation Specifically seeds are extracted from the reads and aligned to the genome with the maximal exact matches These seed alignments are extended with the affine gap Smith Waterman algorithm Page 20 Binom Quick Start
12. e SNP has damaging effect Fathmm tool for predicting the functional effects of protein missense mutations Less score is more pathogenic Prediction D AMAGING for scores less than 1 5 otherwise T OLERATED For isoforms the smallest score most damaging was used Range 16 13 10 64 Phastcons7way vertebrate phylogenetic conservation score based on the multiple alignments log 10 p value The larger the score the more conserved the site Phylop7way vertebrate Phylogenetic conservation score based on the multiple alignments log 10 p value The larger the score the more conserved the site To apply scoring parameters type in a new value next to the score of interest as on Fig 15 and click on Add to filter button Patients Report Q yakovishinad gmail com Filter S cores Add to filter X Depth and quality SIFT Diseases and genes polyphen2 hvar Chromosome position rsID Variant type zygosity effect mutationtaster mutationassessor fathmm cadd phred phastcons way vertebrate phylop7way vertebrate 6 Selecting population frequency Population frequency means how frequent a variant is represented in a population i e a portion of people from a given population sharing the same variant The less frequent a variant is in the population the more chances that the variant is Page 14 Binom Quick Start Guide causative To set the parameters simply type the values in the fields as i
13. e human genome interpretation service iBinom SaaS platform is an end to end genome data analysis solution for diagnostics of Mendelian diseases Unlike typical computer software it is always up to date without the need to install updates Support of non Mendelian diseases tumor profiling and drug response biomarker discovery studies is coming soon iBinom uses Amazon EC2 the world s fastest proprietary algorithms unique machine learning technology and special design of medical report to make inherited diseases diagnostics simpler and faster In addition iBinom has developed an interactive interface clear and explicit for users with a modest bioinformatics expertise it enables clinicians to easily operate the NGS data and detect the causative variants We believe that bringing iBinom service to medicine will solve the diagnostic and treatment odyssey for seriously ill patients This user manual will help you understand the iBinom service better We are constantly updating the manual with the latest explanations on new features and updates to existing functionality You can get the latest version by sending an email to info ibinom com or by simply downloading the manual from the iBinom website We wish you an informative read should you have any further questions on using the iBinom service please get back to us and we will be keen to assist you at any time Page 3 Binom Quick Start Guide Singing up for iBinom account To
14. here to Upload or select from the list below sample fastq gz Page 5 iBinom Quick Start Guide Click on the Patient name to access the Filtering page and to view a list of variants Report av ibinom com Filter Click on to filter Input Data 84576 Q V Create report 84576 variants sample DDXIILI SNP Heterozygote downstream Scores Frequency n 1657A gt G ENST00000456328 rs79585 40 pos 1 14907 DDXIILI SNP To return to the Dashboard from the Filtering page you can always click on the Patients button at the top left side of the page as you see on Fig 3 Page 6 iBinom Quick Start Guide Using iBinom Uploading your data To upload your own sequencing data for analysis click on the Cloud icon see Fig 4 on the left side of the Dashboard or drag and drop your files on it and confirm uploading g BINOM About yakovishinad gmail com New patient Gb of 250 Gb Patients 5 panels 5 exomes 0 genomes Select patient to view and edit report P 4 8 march 2014 sample Drag file here to upload or select from the list below Once uploading has started an arrow indicator of loading process appears in the bottom of the screen At the moment iBinom supports raw sequencing data files in gzip compressed formats e fastq gz e fq gz e vcf gz and uncompressed formats e fastq e fq e bam o vcf e ab1 Note that while both compressed and u
15. l parameters of filtering options press button Add to filter You will be redirected to the interactive filter window see Fig 18 showing the filter adjusted for Clinvar option lt Patients Report O yakovishinad gmail com Filter Click on to filter Q Q Input Data 84576 In the white box a selected filtering option is displayed In the blue box the number of variants matching to your filter parameters is shown The red box displays the number of variants that are not matched You may add the next filtering option at any step for matched unmatched and initial input data by clicking inside the corresponding box To remove a branch click on x inside the white box At the end you may build a tree of the filtering options with you own parameters as shown on Fig 19 Matched x a Matched Panels Depth gt 2 54 x Edit Not matched F 54 clinvar signe X Edit Input Data Not matched Quality x 84576 84522 Edit Not matched F 84522 Matched 700 Panel x Page 16 Binom Quick Start Guide Viewing filter results To see the variants that passed a certain filtration step click on a box of interest the variants will be listed on the page below the interactive filter window as per Fig 20 lt Patients Report yakovishinad gmail com Filter Click on to filter Input Data clinvar_signifie gt X N 576 Edit Q Not matched 556 WY 20 variants sa
16. mple Create report DBT SNP gt Heterozygote non synonymous Scores Frequency exac_amr_af 0 96277 coding MT psC7vr CDDp F MMr PP3V plP7vr SFTr kk_afrk_amrk_eask_eurk_sasae_aae_eaxx_afrx _amrx_adjx_easx_finx_nfex_othx_sast p Ser384Gly c 1 1504 gt G Clinvar ENST000003701 32 rs12021720 Agt Ggt Pathogenic pos 1 100672060 The results comprise a list of variants that satisfy your filtering parameters Fig 21 displays the key elements of an example variant Each variant contains information about its type gene location score ranking from different tools and frequency in populations Each score is represented by a circle on the Scores scale the left side of the scale corresponds to benign and the right to pathogenic scores To view the exact values of each score mouse on the corresponding circles List of frequencies is represented by a right graph Each frequency is represented by a line the greater the angle the greater is the population frequency Mouse on a line or on a population symbol to see the exact frequency value lt Patients Report O yakovishinad gmail com Filter Click on to filter AMPDI SNP Heterozygote stop gained splice site Scores region MT psC7vr CDDp plP7vr kk_afrk_amrk_eask_eurk_sasae_aae_eaxx_afrx amr x_adjx_eas x_finx_nfex_othx_sast p Gin45 c 133C gt T Clinvar ENST000005201 13 rs17602729 Caa Taa Pathogenic pos 1 115236057 Page 17 iBinom Quick Start Guide
17. ncompressed data are accepted uploading of gzip formats takes less time therefore decreasing total amount of time required per an analysis Page Binom Quick Start Guide Once uploading has completed your files are displayed at the left bar of the Dashboard In order to delete the file mouse to the corresponding file name and press the cross icon which will appear on the right side Be careful as this operation cannot be undone You may also download the file to your computer by clicking on the small cloud icon located next to the cross as per Fig 5 BINOM About yakovishinad gmail com New patient Gb of 250 Gb Patients 5 panels 5 exomes 0 genomes i Select patient to view and edit report gt 8 march 2014 sample Drag file here to Upload or select from the list below sample fastq gz ok Starting data analysis To start data analysis follow the instructions and see Fig 6 1 Select a file from the left bar of the Dashboard 2 Press Create a new patient button and confirm beginning of the analysis To see an analysis progress check an indicator at the right side sinom New patient Gb of 250 Gb 5 panels 5 exomes 0 L Create new patient About av ibinom com Patients Select patient to view and edit report Analysing sequencing data sample 280 8 march 2014 sample or Upload more files sample fastq gz Accessing the results and filtering page Once analysis has
18. ndicated on Fig 16 lt Patients Report ri Populations frequency Depth and quality 1000Gp3 Diseases and genes 1000Gp3 afr Chromosome position rsID 1000Gp3 eur Variant type zygosity effect 1000Gp3 amr Scores 1000Gp3 eas Populations frequency 1000Gp3 sas Clinvar ESP6500 aa 7 Selecting database parameters yakovishinad gmail com Add to filter XxX Clinvar is a database that aggregates supporting evidence of relationship among medically important variants and phenotypes Variant classification varies from benign to pathogenic a variant may be classified as of unknown significance when the supporting evidence is weak Tick the box next to a desirable classification parameter as per Fig 17 and click Add to filter Choose a parameter corresponding to American College of Medical Genetics recommendations see http www ncbi nlm nih gov pubmed 25741868 lt Patients Report a Fiker Databases Depth and quality Clinvar Diseases and genes Chromosome position rsID Variant type zygosity effect Scores Populations frequency Mm Databases ACMG Binom Ouick Start Guide Pathogenic Likely pathogenic Benign Likely benign Drug response Histocompatibility Not provided Uncertain significance x Pathogenic Likely pathogenic Benign Likely benign Uncertain ignat 990 gmail com Add to filter X Page 15 General filter use Once you have chosen one or severa
19. ng Results page and included in the iBinom variant report iBinom continue improving the service by updating it with additional databases and features Stay tuned List of the external annotation databases and scores the iBinom service refers to e 1000 Genomes phase 3 15 feb e Clinvar 15 sep e dbSNP build 144 15 jun e dbNSFP v3 0 15 aug e snpEff 4 1b 15 feb e MutationAssessor v2 13 sep e MutationTaster v2 14 jul e ExAC v0 3 dec 14 e ESP6500 14 nov e UK10K 15 jun e PolyPhen hvar v 2 2 2 12 feb e Fathmm v2 3 14 nov e SIFT ensembl 66 15 jan e phastCons 14 jun e phyloP 14 jun e OMIM 15 oct e SiPhy 29way 11 may e GERP 11 may Page 24 Binom Quick Start Guide
20. omal Sex Diseases and genes 2 3 4 5 Chromosome position rsID 6 7 8 9 10 Vari ity eff ariant type zygosity effect 7 12 3 14 15 Scores 16 17 18 19 20 Populations frequency 21 22 X Y M Clinvar Position rsID 4 Selecting variant type zygosity and effect Variant has three main types SNP single nucleotide polymorphism DEL deletion up to 10 15 bases and INS insertion up to 10 15 bases You may choose one or several types at once all types by default Page 12 Binom Quick Start Guide Zygosity a property of the variant that shows if it is homozygous variant is presented on two alleles or heterozygous variant is presented on one allele Effect shows how the variant can effect on the protein behavior HIGH the variant is assumed to have high disruptive impact on the protein probably causing protein truncation loss of function or triggering nonsense mediated decay stop gained frameshift_variant MODERATE a non disruptive variant that might change protein effectiveness missense variant inframe_deletion LOW assumed to be mostly harmless or unlikely to change protein behavior synonymous _ variant MODIFIER usually non coding variants or variants affecting non coding genes when predictions are difficult to make or there is no evidence of impact exon_variant downstream_gene_ variant Select desired parameters by ticking the boxes next to them as per Fig 14 Patients Repo
21. ou have forgotten your password please click on Forgot password on the login window A password reset instructions will be sent to your registered email address Should you have problems with accessing your account at any time please send an email to info ibinom com and our Support team will get back to you as soon as possible Page 4 Binom Quick Start Guide Getting started Once you have logged in to your iBinom account you will see the Dashboard page shown as per Fig 1 o BINOM About av ibinom com New patient Gb of 250 Gb Patients 5 panels 5 exomes 0 genomes s P g Select patient to view and edit report D 8 march 2014 sample Drag file here to upload or select from the list below sample fastq gz Upload file vcf bam fastq S CM A preconfigured dataset called sample is available for you to get acquainted with the service To have an overview of iBinom interface features mouse to sample patient at the Patients list as shown on Fig 2 You will see sample highlighted with red color and various options you may select to proceed with Click on a corresponding link at the right side to download the Quality Control report or variant call file in VCF CSV format for demo data a BINOM About av ibinom com New patient Gb of 250 Gb Patients 5 panels 5 exomes 0 genomes Select patient to view and edit report D 8 march 2014 x Y sample VCF CSV Quality control Drag file
22. realignment is performed to recover indels that occur at the end of a read but appear to be contiguous mismatches Variant calling benchmarking We have benchmarked SAMtools see as iBinom on diagram against the most popular open source variant calling pipelines Bowtie2 GATK and BWA samtools and proprietary DNANexus solution using GCAT service We used Illumina Exome 30x covered NA12878 dataset as we considered it the typical data that one could upload to our solution The dataset is widely perceived to be the gold standard as a part of wet lab and bioinformatics pipeline validation The Venn diagram results are represented on Fig 27 iBinom Bowtie2 Gatk_UG Bwa Samtools DNANexus Variants called by iBinom solution demonstrated the top level of concordance compared to the other pipelines due to applied filtering algorithms High discordance of the variants detected by the other pipelines occurs in some positions and might be explained by varying features and thresholds that variant callers use Because of the low coverage such positions are supposed to produce more errors in variant calling too As soon as for medical applications we seek for the most Page 22 iBinom Quick Start Guide confident variants iBinom prioritizes a higher precision rate over sensitivity and specificity To measure the calling quality we used genotyping data from HumanOmni2 5 8v1 array provided by GCAT server iBinom Bowtie2 Gatk UG Bwa
23. rt yakovishinad gmail com Add to filter xX Depth and quality Filter Var ant YP 7ygos ity vi Diseases and genes Chromosome position rsID Variant type zygosity effect Variant type SNP DEL INS Zygosity Homozygote Heterozygote Effect Modifier Low Moderate High Codon change plus codon deletion Codon change plus codon insertion Codon deletion Codon insertion 5 Selecting scores Scores shows a probability that a variant affects the protein function scores are given to variants by different tools The tools included in the system are SIFT Sorting Intolerant From Tolerant predicts whether an amino acid substitution affects protein function A score lt 0 05 is considered damaging PolyPhen 2 Polymorphism Phenotyping v2 predicts the possible impact of amino acid substitutions on the stability and function of human proteins Probability for a variant being damaging 0 909 1 damaging 0 447 0 908 possibly damaging Page 13 Binom Quick Start Guide 0 0 446 benign MutationTaster employs a Bayes classifier to eventually predict the disease potential of an alteration The probability value is the probability of the prediction i e a value close to 1 indicates a high security of the prediction Mutationassessor predicts the functional impact of amino acid substitutions in proteins such as mutations discovered in cancer or missense polymorphisms The lower the score the more likely th
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