Alamut-HT 1.0 User Manual
Contents
1. Splice site intronic range Intronic variants located within the specified range lt n gt from the nearest splice site are annotated as splice site in the varLocation annotation field ssIntronicRange lt n gt Genes of interest file List of genes of interest A file of HGNC gene symbols 1 per line If this is specified only variants mapped to the listed genes are annotated glist lt gene list file name gt Regions of interest file List of regions of interest ROIs A tabulated file where ROls are described as lt chromosome start end gt BED format Only variants located in ROIs are annotated roilist lt ROI list BED file name gt External annotation file Output parameters Output annotated variants List of external variant annotations to be reported in output format described below Comment By default variants that cannot be annotated are now v 1 1 also extAnnFile lt external annotation file name gt Command line an reported in the annotation output outputannonly Y file This option cancels this behavior Output VCF QUAL field applies to VCF _ SSES VCF input files only outputVCFQuality Output VCF FILTER field applies to SEKR VCF input files only outputVCFFilter VCF information Output VCF INFO fields specified by a list of IDs e g DP AF AA applies to VCF input files only outputVCFInfo ID ID VCF genotype dat2. Updating the alamut_db database To update the alamut db database just download the latest snapshot from http downloads interactive biosoftware com and edit the alamut batch ini file to change the Database File field appropriately Interactive Biosoftware 28 28 February 2015
3. strand 1 1 Variants strand must be explicitly 1 forward strand Strand specified either for the entire A reverse strand input file or on a per variant basis as specified in column 6 of input 0 per variant file default 1 Each variant will be annotated on Annotate variants on all all available transcripts if this oe de alltrans transcripts option is specified Otherwise only the longest transcript is used Not applicable to VCF input G Each variant will be annotated on Annotate on specified Si e the transcript specified on a per spectrans transcript only BEN variant basis as specified in column 7 of input file Annotate variants on File format described below preferred transcripts listed translist in specified file Compute missense Perform Align GVGD MAPP and nomispred predictions SIFT predictions cancels default behavior Compute NNSPLICE predictions Perform NNSPLICE predictions nonnsplice cancels default behavior Interactive Biosoftware 14 28 February 2015 Compute GeneSplicer predictions Alamut Batch 1 4 User Manual Perform GeneSplicer predictions nogenesplicer cancels default behavior Ignore input errors Proceed even if input has invalid entries ignoreInputErrors Exon numbering Simple sequential or custom if available exon numbering exonnums simple custom default simple
4. What is new in version 1 4 E 7 What Was E EE 7 What was new inversion 1 2 cea odone ako kk ka tib kt a te a a a kk e kk ok ak ke Ak 7 What WAS new in version D 8 E e EE 9 Client Server GUI frontend Windows on 9 Client Server command line program Windows and Linux 9 Standalone command line program LINUX only 10 Matten RE EE 11 Using Alamut Batch ccccccccccsssssscccecececsssesseaeeeceesesceeseeeseeeceescsesesssaeaeeeeeesseeseeaseseeeesessseseaaeas 12 GUI frontend Windows only RE 12 Command line program Windows and Linux 13 Software parameters visu kab dacheaxacnssaged n nanan 14 Transcript file fOCMAL E 17 Exter alannotationTileSu E 17 Using a local HGMD Professional database installation 17 OUTPUE eg tie alan kte nal la te ake rd a kap dy a be pak ki ta bag kle sen saba ele kd oka ke ba ae AM e one dok pase aki net ena bay 18 Viewing annotated variants in Alamut Visual 25 Local splicing effect predictions se iii ouka si kod ok sa af ik lk a n fk a ta ai ia rai a piki kai 26 Installing Alamut Batch Standalone ewa on den ak py kit kt kk l a e l kk ka kk e a en 27 Alamut Batch Standalone compone
5. activation is reported as weak If it is greater than 10 then it is reported as strong If at position p there is a significant prediction set on the mutated sequence but not on the wild type sequence then Alamut Batch predicts a new splice site creation Interactive Biosoftware 26 28 February 2015 Alamut Batch 1 4 User Manual Installing Alamut Batch Standalone Alamut Batch Standalone components Alamut Batch Standalone includes the following components 1 The Alamut database It stores all gene related information used by the software 2 The alamut batch program It computes variant annotations based on data provided by the database and results computed by ancillary programs 3 Ancillary programs These are external software tools specialized in computing missense and splicing predictions e g SIFT NNSPLICE The Alamut Database As of version 1 1 11 the Alamut database is supplied as a single compressed file to be used as is by the alamut batch program MySQL is no longer required This file is a snapshot of the live database used by Alamut Visual and the Alamut Batch Client Server version Since the live Alamut database is frequently updated bi monthly snapshots are provided for Alamut Batch Standalone and can be downloaded from the Alamut website The Alamut database includes encrypted gene related information and must be queried by the alamut batch program only The current size of the database is 3 5 GBytes estim
6. maternal etc separated list Possible values clinical testing research literature only etc separated list separated list Number of stars 0 4 separated list DP DFP FP FTV DM DM see HGMD Documentation website separated list separated list separated list separated list transition transversion February 2015 All coding substitutions WT AA 1 letter WT AA 3 letters WT codon WT codon frequency Variant AA 1 letter Variant AA 3 letters Variant codon Variant codon frequency AA Position Missense only Number of orthologues in alignment Number of conserved residues in alignment Most distant species in which AA is conserved BLOSUM45 BLOSUM62 BLOSUM80 WT AA composition Variant AA composition WT AA polarity Variant AA polarity WT AA volume Variant AA volume Grantham distance AlignGVGD class AlignGVGD variation GV AlignGVGD deviation GD SIFT prediction SIFT weight SIFT median Interactive Biosoftware Alamut Batch 1 4 User Manual wtAA_1 wtAA_3 wtCodon wtCodonFreq varAA_1 varAA_3 varCodon varCodonFreq posAA nOrthos conservedOrthos conservedDistSpecies BLOSUM45 BLOSUM62 BLOSUM80 wtAAcomposition varAAcomposition wtAApolarity varAApolarity wtAAvolume varAAvolume granthamDist AGVGDclass AGVGDgv AGVGDgd SIFTprediction SIFTweight SIFTmedian 23 28 Februa
7. 4 User Manual Local splicing effect predictions Alamut Batch interprets raw splice site signal recognition by MaxEntScan NNSPLICE and Human Splicing Finder HSF in the variation vicinity to provide predictions about the creation of new splice sites or the activation of existing cryptic sites Note that this is different from predictions at the nearest splice site where only raw prediction scores are provided but not interpreted by Alamut Batch This section describes how local splicing effect predictions are computed Only the MaxEntScan NNSPLICE and HSF splice site predictors are used in the interpretation algorithm The following thresholds are used to consider or discard raw predictions e A MaxEntScan score is deemed significant if gt 0 e An NNSPLICE score is deemed significant if gt 0 4 e AnHSF score is deemed significant if gt 60 Let s define a raw prediction set as a set of raw predictions at the same position for the same signal A raw prediction set is deemed significant if at least two of MaxEntScan NNSPLICE or HSF predictions are significant If at position p excluding natural splice site positions there is a significant prediction set both on the wild type sequence and on the mutated sequence and if the mutated prediction set is significantly higher than the wild type then Alamut Batch predicts a cryptic splice site activation If the change is less than 3 it is not reported If it is less than 10 then the
8. Alamut Server name in field Network IBS Server is a ht interactive Interactive Biosoftware 9 28 February 2015 Alamut Batch 1 4 User Manual biosoftware com by default If you are based in North America please change the server name to a ht na interactive biosoftware com Standalone command line program Linux only See Installing Alamut Batch Standalone at the end of this document Interactive Biosoftware 10 28 February 2015 Alamut Batch 1 4 User Manual Variant Input file The software takes on input a list of genomic variations and outputs a list of annotations for each variant when it is located on a gene available in the Alamut database Alamut Batch supports VCF files and tab delimited files on input VCF files This is the most common format for variant description Alamut Batch supports VCF v4 0 and later Note that variants are implicitly processed on the forward strand and that monomorphic references i e entries with no alternate alleles are not supported Tab delimited files A specific tab delimited text format can also be used for variant input In this format each line should contain the following fields separated by tab characters 1 Variant id anything Chromosome 1 22 X Y Genomic position Reference nucleotide s ACGT or for insertions Mutated nucleotide s ACGT or for deletions Optional strand 1 or 1 used if strand parameter is set to 01 Optional transc
9. be restricted to a list of preferred transcripts specified in a gene transcripts file translist option e Annotation can also be restricted to a range of variants of the input file from and to options not available in the Windows GUI Two other new features are specific to the Standalone version e Multi process support Annotation jobs can now be split among multiple processes on the same computer processes option e Access to local HGMD Professional database installations has been changed since BIOBASE no longer provides a query API see Using a local HGMD Professional database installation Interactive Biosoftware 7 28 February 2015 Alamut Batch 1 4 User Manual What was new in version 1 1 Here are the new features introduced in version 1 1 Integration of HGMD the Human Gene Mutation Database data available to HGMD Professional subscribers Integration of NHLBI GO Exome Sequencing Project ESP data Unannotated variants are now reported in the annotation output file and in the failed variants output file as well unless the outputannonly option is specified If the new option ssIntronicRange n is used intronic variants located within the specified range n from the nearest splice site are annotated as splice site in the varLocation annotation field Variants can now be filtered by regions of interest defined in a BED format file roilist lt ROI list BED file name gt External annotatio
10. Ki INTEractive biosoftware alanqut A BATCH 14 user manual Alamut Batch 1 4 User Manual This document and its contents are proprietary to Interactive Biosoftware They are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in anyway whatsoever without the prior written consent of Interactive Biosoftware Interactive Biosoftware does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s 2015 Interactive Biosoftware All rights reserved Interactive Biosoftware 2 28 February 2015 Alamut Batch 1 4 User Manual Contents EEN 5 Product descriptio EE 5 Standalone VEN E ON kq aa bn kdk naj asa k en pous ko k kt ken kaa a tas a bite la ra anna eaae ritear 5 Client Server version 5 System regue En EE 6 Standalone VENOM bois pa bs vs pk asasen e kk pak ke eka k a a n vi RA kak eks a ka ka AA 6 Client Server version 6
11. Python MySQLdb package Interactive Biosoftware 17 28 February 2015 Alamut Batch 1 4 User Manual Output The output of Alamut Batch is a tab separated file of annotations 1 line per variant or multiple lines per variant if annotation is performed on multiple transcripts Annotations produced are listed below User defined input fields are reported as is in the last output columns NOTE SA The Chromosome field chrom was previously the fifth output field As of v1 2 it now comes as the second field followed by the new Variant position field pos that replicates the original variant position given in the input file Annotation Name Comment Id Id Variant id as supplied in input file Chromosome chrom Variant position pos As supplied in input file Failed annotation reason unnnotatedReason Field not available if option outputannonly is used Gene symbol gene HUGO Gene Nomenclature Committee HGNC symbol Gene id HGNC geneld HGNC id Transcript transcript e g NM_000249 3 Transcript strand strand Transcript length transLen Full cDNA length Protein protein e g NP_000240 1 Uniprot Uniprot Uniprot accession e g P40692 Variant Type varType substitution deletion insertion duplication delins Variant coding effect codingEffect synonymous missense nonsense in frame frameshift start loss stop loss Variant location varLocation upstream 5 UTR exon intron 3 UTR downstream splice site see s
12. a Output VCF genotype fields specified by a list of IDs e g GT AC GQ applies to VCF input files only outputVCFGenotypeData ID ID Empty values HGMD parameters Interactive Biosoftware Empty output fields are populated with specified value e g NULL Comment 15 28 outputEmptyValuesAs lt value gt Command line February 2015 HGMD Professional login Proxy parameters Internet proxy options Alamut Batch 1 4 User Manual Comment hgmdUser lt HGMD Pro user name gt hgmdPasswd lt HGMD Pro password gt Command line proxyserver lt proxy server name gt proxyport lt proxy server port number gt proxyuser lt proxy user login gt proxypasswd lt proxy password gt Interactive Biosoftware 16 28 February 2015 Alamut Batch 1 4 User Manual Transcript file format The input file for preferred transcripts is tab delimited and requires at least two columns gene name and transcript name Multiple transcripts per gene can be specified in additional columns as in the following example BRCAI gt NM 007294 3 MLH1 gt NM_000249 3 gt NM_001167618 1 External annotation files External variant annotations e g variant pathogenicity status as previously established in the lab can be integrated in the annotation output Variants are described using the chromosome name and genomic level nomenclature Variants and annotations
13. ated growth 3 GBytes year Software Programs All the required programs are either Linux executables or Python 2 6 scripts They must all be installed on the same Linux computer Ancillary programs include missense and splicing prediction tools that are either provided with the Alamut Batch Standalone package or can be installed separately see below System Requirements See above Installing Installing Alamut Batch Standalone requires two steps e Installing the alamut_db database e Installing software components Alamut Batch and ancillary programs Installing the alamut_db database Go to the Alamut Batch Standalone section of http downloads interactive biosoftware com and download the latest database snapshot Place the donwload file anywhere in the local filesystem of the computer running Alamut Batch Installing Alamut Batch Go to the Alamut Batch Standalone section of http downloads interactive biosoftware com and download the latest tarball Edit the alamut batch ini file and supply Interactive Biosoftware 27 28 February 2015 Alamut Batch 1 4 User Manual e Your Institution ID in the Institution field e Your license key in the Licence Key field e User initials as appropriate in the User field _ NOTE v The Alamut Server name in field Network IBS Server is a ht interactive biosoftware com by default If you are based in North America please change the server name to a ht na intera
14. ctive biosoftware com Set the Database File field to the full path of the downloaded database file Installing ancillary programs All ancillary software programs must be installed in the alamut batch standalone ancillary directory gt cd alamut batch standalone ancillary SIFT Download and uncompress gt wget http sift jcvi org www sift4 0 3b tar gz gt tar zxf sift4 0 3b tar gz MAPP optional Download file MAPP zip from http downloads interactive biosoftware com Linux Section Alamut Batch Standalone gt Other Downloads Unzip this file inside the ancillary sub directory NNSPLICE optional Obtain package NNSPLICEO 9 from Martin Reese mreese omicia com and unpack in the ancillary directory Note that NNSPLICE requires glibc i686 GNU 32 bit libc library Other prediction tools Other tools are either provided with the Alamut Batch distribution GeneSplicer and MaxEnt or are embedded inside al amut batch Align GVGD SSF HSF Python proxy programs Two Python proxy programs are needed to ease the communication between Alamut Batch and the ancillary programs mispred_ht py and nnsplice_ht py Both are provided in the Alamut Batch distribution and must reside in the ancillary directory The getHGMD py program also provided in the Alamut Batch distribution serves as a proxy to connect to a local HGMD Professional database if any This program requires the MySQLdb Python package
15. g shows where Alamut Batch and Alamut Visual take place in a typical NGS analysis pipeline DNA Reads Variants Mutations m_ S SO SO Sequencer Alignment venient Annotation Filterin Iinternictation cd 8 Calling 8 Reporting alamut alamut d batch d visual lt Alamut Batch can be used independently from Alamut Visual However results from Alamut Batch can be easily injected into Alamut Visual so as to benefit from its rich feature set including graphical visualization Alamut Batch annotates variants by querying a database storing information about human genes the Alamut database Technically Alamut Batch comes in two versions depending on where the gene database is located e The Standalone version uses a locally installed database e The Client Server version connects over the internet to our hosted database Standalone version The Standalone version of Alamut Batch provides best performance by including in a local installation all software components and the Alamut database required by the annotation process It is most appropriate for intensive variant annotation needs such those of whole exome analyses Alamut Batch Standalone is a Linux command line program Client Server version The Client Server version of Alamut Batch connects remotely to the central Alamut database Due to internet latency the Client Server version is slower than the Standalone Interactive Biosoftware 5 28 Februar
16. he nth variant assbly NCBI36 GRCh37 GRCh38 default GRCh37 strand 1 1 default 1 per variant not applicable to VCF input alltrans annotate variants on all transcripts spectrans annotate variants only on specified per variant Transcripts not applicable to VCF input 1 2 translist transcript file name gt annotate variants only on listed preferred transcripts glist lt gene list file name gt list of genes of interest roilist lt ROI list BED file name gt list of regions of interest nomispred no missense predictions faster nonnsplice no NNSPLICE predictions faster nogenesplicer no GeneSplicer predictions faster ignoreInputErrors proceed even if input has incorrect entries exonnums simple custom default simple ssIntronicRange lt n gt set varLocation as splice site if variant is intronic and within this range extAnnFile lt external annotation file name gt include additional annotations from external file outputannonly output only annotated variants in annotation output outputVCFQuality outputVCFFilter outputVCFInfo ID ID outputVCFGenotypeData ID ID outputEmptyValuesAs lt value gt e g NULL hgmdUser lt HGMD Pro user name gt hgmdPasswd HGMD Pro password gt proxyserver lt proxy server name gt proxyport proxy server port number gt proxyuser lt proxy user login gt p
17. n American population Alternate allele frequency in African American population Alternate allele frequency in all populations Interactive Biosoftware Alamut Batch 1 4 User Manual exacAFRHmz exacAMRHmz exacEASHmz exacSASHmz exacNFEHmz exacFINHmz exacOTHHmz exacFilter exacReadDepth espRefEACount espRefAACount espRefAllCount espAltEACount espAltAACount espAltAllCount espEAMAF espAAMAF espAllMAF espEAAAF espAAAAF espAllAAF 21 28 February 2015 Average sample read depth ClinVar ids ClinVar origins ClinVar methods ClinVar clinical significances ClinVar review status ClinVar phenotypes HGMD mutation id HGMD phenotype HGMD PubMed id HGMD sub category COSMIC ids COSMIC tissues COSMIC frequencies COSMIC sample counts Indels Inserted nucleotides Deleted nucleotides Substitutions Type WT nucleotide Variant nucleotide Nucleotide change PhastCons score phyloP Interactive Biosoftware Alamut Batch 1 4 User Manual espAvgReadDepth clinVarlds clinVarOrigins clinVarMethods clinVarClinSignifs clinVarReviewStatus clinVarPhenotypes hgmdld hgmdPhenotype hgmdPubMedld hgmdSubCategory cosmiclds cosmicTissues cosmicFreqs cosmicSampleCounts insNucs delNucs substType wtNuc varNuc nucChange phastCons phyloP 22 28 separated list separated list Possible values germline somatic de novo
18. ns supplied in variant annotation files can now be integrated in the output extAnnFile lt external annotation file name gt Version 1 1 3 adds three output fields reporting validation details of dbSNP entries Version 1 1 3 also adds three output fields reporting frequencies of ESP alternate alleles alternate alleles not always being minor alleles Version 1 1 4 adds an option to allow processing even if the input file has invalid entries Version 1 1 5 fixes a bug where variants affecting multiple genes where not processed on all genes Version 1 1 6 adds the HGMD variant sub category output field and fixes a network proxy bug for HTTPS Version 1 1 7 brings improvements to the GUI version all command line options are now also available in the graphical interface With version 1 1 7 it is now possible to input variant alleles that are the same as the transcript allele e g when the genome reference sequence has the minor allele of a SNP and the transcript has the major allele Version 1 1 7 can query a local HGMD database installation Version 1 1 8 fixes a bug occurring when a gene cannot be loaded Version 1 1 9 features performance improvements and support for mitochondrial variants Version 1 1 10 fixes a bug causing software crashes on transcripts where the STOP codon is isolated in a 3 UTR exon Version 1 1 11 supports a wider range of VCF variant descriptions i e descriptions that don t strictly comply with the format specificati
19. nts 27 The Alamut KEE E 27 Software Drograms ENER 27 System Requirements viiesa aana a aaa a aaa aaa taa Uaes iaa iaia 27 Interactive Biosoftware 3 28 February 2015 Alamut Batch 1 4 User Manual ee EE ER Installing the alamut db databasE vvrttttttetteteeeeesoooooooessesesseoooooooossasensaoonosooooensonn 27 Installing Alamut Batch 27 Installing ancillary progra MS EE 28 Oth r prediction tools EE 28 Python Proxy Drograms ccccsessssscececeeecsessnseceeeceeecseseseaeeeeeeeesseeseaeeeseceesseeseaeaeeeeeesensees 28 Updating the alamut_ Databases Genee Geheit 28 Interactive Biosoftware 4 28 February 2015 Alamut Batch 1 4 User Manual A propos This user manual describes how to install and use Alamut Batch version 1 4 0 Feb 2015 _ NOTE s Alamut Batch was previously named Alamut HT Product description Alamut Batch is a high throughput annotation engine for NGS analysis Designed for intensive variant analysis workflows this software enriches raw NGS variants with dozens of annotations including effects on human genes detailed SNP information and missense and splicing predictions Annotations provided by Alamut Batch are similar to those available in the Alamut Visual mutation interpretation software Alamut Batch is able to annotate tens of thousands variants per hour This schematic drawin
20. on ExAC allele frequency in other populations Interactive Biosoftware Alamut Batch 1 4 User Manual rsValidations e g Cluster Frequency 1000G rsValidationNumber rsAncestralAllele rsHeterozygosity rsClinicalSignificance rsMAF rsMAFAllele rsMAFCount 1000g_AF 1000g_AFR_AF 1000g_SAS_AF 1000g_EAS AF 1000g_EUR_AF 1000g AMR AF exacAllFreq exacAFRFreq exacAMRFreq exacEASFreq exacSASFreq exacNFEFreq exacFINFreq exacOTHFreq 20 28 February 2015 ExAC homozygosity ratio in African population ExAC homozygosity ratio in Latino population ExAC homozygosity ratio in East Asian population ExAC homozygosity ratio in in South Asian population ExAC homozygosity ratio in Non Finnish European population ExAC homozygosity ratio in in Finnish European population ExAC homozygosity ratio in other populations ExAC VCF filter value ExAC read depth ESP reference allele counts in European American population ESP reference allele count in African American population ESP reference allele count in all populations ESP alternate allele count in European American population ESP alternate allele count in African American population ESP alternate allele count in all populations Minor allele frequency in European American population Minor allele frequency in African American population Minor allele frequency in all populations Alternate allele frequency in Europea
21. on and can now output VCF genotype fields of all input samples Interactive Biosoftware 8 28 February 2015 Alamut Batch 1 4 User Manual Installation Download the software from http downloads interactive biosoftware com The downloaded file is a self extractable archive on Windows and a tarball on Linux Extract the contents Client Server GUI frontend Windows only Launch the program Alamut Batch Ul exe Open the Option panel and supply e Your Institution ID in the Institution field e Your license key in the Licence Key field e User initials as appropriate in the User initials field If your internet access is behind a proxy you will also need to supply appropriate proxy settings NOTE ta The Alamut Server name is a ht interactive biosoftware com by default If you are based in North America please change the server name to a ht na interactive biosoftware com r 7 Options User Insttuson Id License Key User initials HGMD User Password Network Alamut Server a ht interactwe tiosoftware com Server Port 30 Use proxy server Proxy Server Proxy Port Alamut Batch Version 1 3 Rev 0 Le Cancel Client Server command line program Windows and Linux Edit the alamut batch ini file and supply e Your Institution ID in the Institution field e Your license key in the Licence Key field e User initials as appropriate in the User field D NOTE v The
22. otations from these datasets are output as lists where each item is separated by a character Lists in each field are ordered by dataset entries For example variant MLH1 NM_000249 2 c 793C gt T has 3 entries in ClinVar yielding the following ClinVar annotation fields RCV000022502 22 RCV000075872 1 RCVO00034802 1 clinVarlds clinVarOrigins germline germline germline clinVarMethods literature only research research Pathogenic Pathogenic VUS 11311 clinVarClinSignifs clinVarReviewStatus clinVarPhenotypes Lynch syndrome ii Lynch syndrome Not provided What was new in version 1 3 Version 1 3 adds support for the GRCh38 hg38 human genome assembly and includes 1000 genomes Phase 3 version 5 variant frequencies for five sub populations African East Asian South Asian European American What was new in version 1 2 Version 1 2 introduces the following new features e Support for non protein coding genes now available in the Alamut gene database e Output annotation lines now include the original variant position provided in the input variant file This helps in reconciling variants between the output annotation file and other variant files which could previously show problematic in case of variant position changes due to application of HGVS rules e VCF quality filter information and genotype fields are now reported in the output even for not annotated variants e Annotation can now
23. p Leu123Leu Nearest exon if intronic variant 5 3 Predictions at nearest splice site ditto ditto ditto ditto ditto ditto ditto ditto ditto Average change predicted by MaxEntScan NNSPLICE and HSF New Donor Site New Acceptor Site Cryptic Donor Strongly Activated Cryptic Donor Weakly Activated Cryptic Acceptor Strongly Activated Cryptic Acceptor Weakly Activated see Section Local splicing effect predictions yes no yes no Variant flagged as suspect by dbSNP February 2015 dbSNP validation labels dbSNP number of validation categories dbSNP ancestral allele dbSNP variation average heterozygosity dbSNP variation clinical significance dbSNP variation global Minor Allele Frequency dbSNP variation global minor allele dbSNP variation sample size 1000 genomes global allele frequency 1000 genomes allele frequency in African population 1000 genomes allele frequency in South Asian population 1000 genomes allele frequency in East Asian population 1000 genomes allele frequency in European population 1000 genomes allele frequency in American population ExAC global allele frequency ExAC allele frequency in African population ExAC allele frequency in Latino population ExAC allele frequency in East Asian population ExAC allele frequency in South Asian population ExAC allele frequency in Non Finnish European population ExAC allele frequency in Finnish European populati
24. ript id used if spectrans parameter is specified Optional user defined fields e g heterozygosity number of reads etc These fields are not processed but merely reported as is in the output file Sot Se ae Empty lines and lines starting with a character are ignored Example 1d00011 1 23456 T A 42 T gt A substitution id00022 9 876543 TGA 84 TGA insertion 1d00032 5 613720 AC 2 AC deletion 1 Strand is related to the variant itself not to the transcript orientation Interactive Biosoftware 11 28 February 2015 Alamut Batch 1 4 User Manual Using Alamut Batch GUI frontend Windows only Launch the program Alamut Batch Ul exe Program options are spread over three different tabs FI Output annotated variants orty Outpt empty valves as vo FI Output VEF QUAUty field F Outpt VCF FILTER fek Output YO IN Ormaton fiekds DP AF AA Output VCF genotype fields e g GT AC GQ Options are described in section Software Parameters below Interactive Biosoftware 12 28 February 2015 Alamut Batch 1 4 User Manual Command line program Windows and Linux Synopsis alamut batch help listgenes lt output file name NCBI36 GRCh37 GRCh38 in lt variant file name gt ann lt annotation file name gt unann lt unannotated log file name gt from lt n gt start annotating from the nth variant to lt n gt annotate up to t
25. roxypasswd proxy password gt processes lt processes gt Standalone version only k ka N N Options flagged as 1 2 were new in version 1 2 Using the listgenes option puts the program in a special mode making it ouput the unsorted list of genes available in the Alamut database for the given genome assembly Options are described in section Software Parameters below Interactive Biosoftware 13 28 February 2015 Software parameters Input Output files Variant file Alamut Batch 1 4 User Manual Comment Variant input file full path name refer to Section Input file for details of the file format Command line in lt variant file name gt Annotation file Annotation output file full path name refer to Section Output file for details of the file format ann lt annotation file name gt Failed variants file Annotation parameters Output log file name This file lists the variants that could not be annotated Comment Not available in the Windows GUI unann lt unannotated log file name gt Command line from lt n gt start annotating from the nth variant R azi to n annotate up to the nth variant NCBI36 hg18 or GRCh37 hg19 The NCBI36 hg18 genome Sg assembly is still supported but assbly NCBI36 GRCh37 Y youare strongly encouraged to default GRCh37 provide the software with GRCh37 hg19 variations Not applicable to VCF input
26. ry 2015 Alamut Batch 1 4 User Manual MAPP prediction MAPPprediction MAPP p value MAPPpValue MAPP p value median MAPPpValueMedian Interactive Biosoftware 24 28 February 2015 Alamut Batch 1 4 User Manual Viewing annotated variants in Alamut Visual The genomic level and cDNA level HGVS descriptions generated by Alamut Batch annotations gNomen and cNomen can be easily copied and pasted into Alamut Visual Copy a list of HGVS descriptions L o mg o o s gDNAend gNomen cDNAstart cDNAend cNomen 37059009 Chr3 GRCh37 g 37059009A gt G 803 803 NM_000249 3 c 803A gt G 37059031 Chr3 GRCh37 g 37059027_37059030dup 821 824 NM 000249 3 c 821 824dup 37059078 Chr3 GRCh37 g 37059078del 872 872 NM 000249 3 c 872del 41276113 Chr17 GRCh37 g 41276113T gt G 1 1 NM_007300 3 c 1A gt C 41276082 Chr17 GRCh37 g 41276081 41276082insG 32 33 NM 007300 3 c 32 33insC 41276046 Chr17 GRCh37 g 41276045 41276046del 68 69 NM 007300 3 c 68 69del Ctrez sS Chr17 GRCh37 g 41276045_41276046del E Ctrl Y Ctrl X Paste Ctrl V Delete Select All Ctrl A Variants then show up in a variant list Double click on an entry to jump from a variant to another i chr3 GRCh37 g 37059009A gt G chr3 GRCh37 g 37059027_37059030 chr3 GRCh37 g 37059078del chr17 GRCh37 g 41276113T gt G chr17 GRCh37 g 41276081_4127608 chr17 GRCh37 g 41276045_4127604 Interactive Biosoftware 25 28 February 2015 Alamut Batch 1
27. sIntronicRange option Genome assembly assembly gDNA start gDNAstart gDNA end gDNAend HGVS genomic level nomenclature gNomen e g Chr3 GRCh37 g 37059009A gt G cDNA start cDNAstart cDNA end cDNAend HGVS cDNA level nomenclature cNomen e g NM_000249 3 c 803A gt G Interactive Biosoftware 18 28 February 2015 HGVS protein level nomenclature Alt Protein level nomenclature Exon Intron OMIM id Distance to nearest splice site Nearest splice site type WT seq SpliceSiteFinder score WT seq MaxEntScan score WT seq NNSPLICE score WT seq GeneSplicer score WT seq HSF score Variant seq SpliceSiteFinder score Variant seq MaxEntScan score Variant seq NNSPLICE score Variant seq GeneSplicer score Variant seq HSF score Nearest splice site change Splicing effect in variation vicinity Protein domain 1 Protein domain 2 Protein domain 3 Protein domain 4 dbSNP variation dbSNP validated variation dbSNP suspect variation Interactive Biosoftware Alamut Batch 1 4 User Manual pNomen alt_pNomen exon intron omimld distNearestSS nearestSSType wtSSFScore wtMaxEntScore wtNNSScore wtGSScore wtHSFScore varSSFScore varMaxEntScore varNNSScore varGSScore varHSFScore nearestSSChange localSpliceEffect proteinDomain1 proteinDomain2 proteinDomain3 proteinDomain4 rsid rsValidated rsSuspect 19 28 e g p Glu268Gly Like pNomen except for synonymous variants e g
28. should be supplied in tab delimited text files using the following format e First line Tab separated list of annotation labels preceded by chrom and gNomen for clarity For example chrom gt gNomen gt Class gt Freq where gt denotes tabulation characters and Class and Freq are annotation labels e Other lines Tab separated variant description and annotation values in the same order as specified in line 1 For example chr1 gt g 45800167G gt A gt Likely pathogenic gt Q 09Q1 chr13 gt g 32929387T gt C gt Unknown gt 0 005 Annotation labels as supplied in first line are reported in the first line of the output file When input variants and externally annotated variants match the annotation output contains corresponding annotation values Note that multiple external variant annotation files can be supplied using option extAnnFile multiple times Using a local HGMD Professional database installation If you have a downloaded version of HGMD Professional you can let Alamut Batch query it locally rather than over the internet To achieve this you will need to edit the alamut batch ini file and add an HGMD section to specify how to connect to the local server as shown in the following example HGMD host 192 168 0 1 user my_hgmd_user password my_hgmd_passwd database hgmd_pro NOTE v Querying a local H6MD Pro database is available on Linux only and requires Python and the
29. y 2015 Alamut Batch 1 4 User Manual version but is very easy to install It is however an efficient solution for moderate variant annotation needs such those of gene panels sequencing analyses Alamut Batch Client Server is available as a command line program on Windows and Linux operating systems The software is also available with a GUI frontend on Windows System requirements Standalone version Alamut Batch Standalone requires the following system specifications e 64 bit CentOS 6 4 distribution or other compatible Linux distribution e Python 2 6 or 2 7 e Python MySQLdb package if access to a local HGMD Professional database installation is intended e OpenSSL client libraries e g RPM package openssl x86_ 64 e 4GBRAM minimum e 5 GB hard drive space e Internet connection required for license control Client Server version Alamut Batch Client Server requires the following system specifications e 64 bit CentOS 6 4 distribution or other compatible Linux distribution e 4GBRAM minimum e 100 MB hard drive space e Internet connection required e Windows XP 7 or 8 32 bit or 64 bit e 2GBRAM minimum e 50 MB hard drive space e Internet connection required Interactive Biosoftware 6 28 February 2015 Alamut Batch 1 4 User Manual What is new in version 1 4 Version 1 4 adds annotations from ClinVar COSMIC and ExAC Note that since a given genomic variant can match multiple ClinVar or COSMIC records ann
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