As exome sequencing of an individual typically generates thousands of gene variants relative to the human reference sequence, identifying the variant(s) of potential causative effect, i.e. clinically significant variant(s) directly related to the patient’s phenotype, is a challenging and demanding task. The rapid adoption of clinical NGS testing has brought to the forefront the complexities associated with variant interpretation and new important challenges in the clinical interpretation of DNA variants associated with genetic diseases have emerged.
In order to address these challenges, and starting in early 2012, the first version of the Exome Management Application-EMA® pipeline software (v.220.127.116.11) was designed and implemented. The ClinGenics Exome Management Application-EMA® pipeline software is a multi-parametric, multi-stage, phenotype-driven, bioinformatics tool, providing cutting-edge decision support for clinical interpretation of variants generated through applications of Next Generation Sequencing (NGS), involving primarily Whole Exome Sequencing (WES), but also multi-gene and single-gene NGS analysis. For more than 5 years the ClinGenics Exome Management Application-EMA® pipeline has been methodically developed, to become a very powerful variant interpretation tool aiding decisively in the diagnosis of hundreds of complex and rare disorders (see publications). The current version of the Exome Management Application-EMA® pipeline software is at v.18.104.22.168 and is constantly updated.
The final variant interpretation report provided by ClinGenics has the added important feature of incorporating expert manual curation, personalized interpretation and case-specific comments and suggestions for further actions, thus fulfilling its role as a true decision-support tool.
The pipeline is intended exclusively for clinicians and genetics professionals who need a tested and reliable advisory tool to aid in their clinical investigations and diagnosis of patients subjected to clinical exome sequencing studies.
Brief Description of Services
ClinGenics Ltd, through its proprietary Exome Management Application- EMA® pipeline software currently provides the following type of variant data interpretation analysis services:
- Whole Exome Sequencing Trio (WES-trio) parents and proband variant interpretation
- Whole Exome Sequencing (WES) single proband variant interpretation
- Multi-gene NGS panel (50-150 genes) exome sequencing variant interpretation
- Multi-gene NGS panel (1-49 genes) exome sequencing variant interpretation
- BRCA1 and BRCA2 genes NGS panel exome sequencing variant interpretation
- CFTR gene NGS sequencing variant interpretation
For large scale projects or other research applications, a dedicated custom variant analysis is also available for generating population-specific common variant database(s). The same type of custom variant analysis may also be performed and may be useful for identifying common disease-related variants in multiple samples (disease cohorts). The results are made available as a database in SQL file format or custom report may be generated.
Local installations of the software for large-scale projects may be exceptionally considered for non-profit academic and research institutions only.
Please direct your inquiries to email@example.com.
Supported NGS data and platforms
Currently, the ClinGenics Exome Management Application- EMA® pipeline software supports NGS data, in VCF file(s) format version 4.0 or later, generated by:
- Thermo Fisher AmpliSeq library (whole exome or custom panels) and Hi-Q sequencing on Thermo Fisher Ion PGMTM, Ion S5TM, Ion S5TM XL and Ion ProtonTM .
- Exome capture/enrichment (all major exome capture or enrichment suppliers, whole exome or custom panels) and NGS on Illumina MiniSeq TM, MiSeq TM, NextSeq TM or HiSeq TM
Brief description of the Exome Management Application- EMA® pipeline workflow
As the services provided through the ClinGenics Exome Management Application- EMA® pipeline software are intended exclusively for clinicians and genetics professionals, ClinGenics Ltd. reserves the right to deny the creation of an account by a user who does not fulfill the necessary requirements.
Pricing inquiries may be submitted once the user account has been activated
- The user must then upload raw un-annotated VCF file(s) (version 4.0 or later), which are then annotated through a custom annotation pipeline. Typically, raw VCF files (raw variant data) are generated either through the ‘variant caller’ plug-in of the Ion Torrent Server (for Thermo Fisher Ion PGMTM, Ion S5TM, Ion S5TM XL and Ion ProtonTM users, see manufacturer’s instructions) or through the Illumina BaseSpace application (for Illumina MiniSeqTM, MiSeqTM, NextSeqTM or HiSeqTM series users, see manufacturer’s instructions).
- The user must specify the type of NGS platform and reagent used for each type of analysis and select the type of NGS variant interpretation analysis required for the particular case
- The user must complete all the required standardized clinical information in the provided Data Submission Form (anonymised, without any patient identifiers). Other relevant case-related data may be included and they will be incorporated into the analysis pipeline accordingly. ClinGenics does not request and will not accept data files or case-related clinical information which may contain patient-specific identifiers (e.g. name, address, etc.).
- Upon uploading the vcf file(s), the case-related clinical information and other required data files, the user is notified by email: (a) that the uploaded data were successfully submitted and also (b) the expected time of completion of the requested type of analysis.
- The submitted un-annotated vcf file is annotated using a customized annotation workflow (see demo report and supporting files).
- The annotated vcf file is subsequently imported into the ClinGenics Exome Management Application-EMA® pipeline, our powerful and tested bioinformatics platform-software pipeline, coupled to expert curation for the evaluation and reporting of clinically actionable genomic variants, taking into account the user-provided sample-specific information as stated in the data submission form.
- Duplicate variants are removed. Remaining variants are initially filtered-out based on curated information maintained in the pipeline relating to platform-specific known recurrent NGS and variant calling artifacts (known recurrent false-positives related to the specific sequencing platform/chemistry, derived from whole exome sequencing samples analyzed on the Thermo Fisher and Illumina NGS platforms).
- All remaining variants are then assigned an EMAPathScore, derived through a built-in multi-parametric in silico pathogenicity score prediction algorithm.
NOTE: Out of ~28.000 curated pathogenic coding and ± 6bp splice-site variants (according to ClinVar or HGMD), EMAPathScore has correctly categorized 97.8% of these as pathogenic or likely pathogenic (~98% concordance).
- Using the ClinGenics Exome Management Application-EMA® Disease Relevance (EMADR®) pipeline module, variants are subsequently prioritized in terms of their clinical relevance and mode of disease inheritance, based on the submitted diseases, disease phenotypes and other relevant clinical information supplied by the user.
- Variants are then further evaluated/prioritized through the Exome Management Application-EMA® Variant Database (EMAVAR®) pipeline module based on information relating to: (a) their presence and frequency in international databases (1000GP, NHLBI Exome Variant Server, ExAc, GnomAD), (b) their pathogenicity as reported in various disease/gene specific variant/mutation databases (e.g. ClinVar, HGMD, etc.) and (c) clinical information and annotation maintained in the EMAVAR® pipeline database from previously diagnosed cases (currently ~120 million annotated variants).
- Finally, remaining variants are curated by the ClinGenics team based on expert clinical knowledge and according to published guidelines (ACMG-AMP guidelines).
- Upon completion of variant interpretation analysis the user is notified by email and is then able to login securely and retrieve-download the final variant interpretation report(s) and accompanying supporting data files. The original vcf data file(s) submitted for analysis may then be deleted.
For a full step-by-step description of the data submission process, please consult the Exome Management Application-EMA® Workflow Manual (available upon registration). Also, upon registration a user can download and view a Demo Report together with the supporting data files.
What is the time required for analysis and report generation?
Taking into account world time differences, analysis and final variant clinical interpretation of whole exome sequencing data is typically completed within 12–24 hours, depending on the type of case-related clinical parameters. Analysis and final variant clinical interpretation of data involving NGS multi-gene panels, BRCA1/ BRCA2 genes and CFTR gene is typically completed in less than 12 hours. If a report is not issued within the expected time, the user is notified by email as to the reason of the delay and the expected time of completion.
Upon completion, the user receives an email notification and may login and retrieve/download the final report together with the accompanying data files.
Pricing Structure and Policy
A free annual-12 month (renewable) user subscription/registration for use of ClinGenics services is required.
Pricing inquiries may be submitted once a user account has been activated.
In order to be able to submit data files for analysis, the user must first submit a price inquiry and select the payment options. Pre-payment may be required prior to submission.
The user is prompted to select the type of variant interpretation service(s) and the number of data files he wishes to submit for analysis. The user has the option to purchase a one-time submission of one or more data files, or to purchase in advance any number of data file submissions. A discount applies depending on the number of data files submitted or purchased in advance.
ClinGenics will issue the relevant invoice according to the billing address provided by the user.
The ClinGenics Exome Management Application-EMA® pipeline software is For Research Use Only. Not for use in diagnostic procedures.
Specifically, the variant interpretation services provided by ClinGenics Ltd. (ClinGenics) are intended for RESEARCH USE ONLY (RUO) and should not be regarded as clinical or medical in nature.
By submitting Next Generation Sequencing (NGS) data to ClinGenics the user acknowledges that the results of variant interpretation, as delivered, DO NOT constitute a diagnosis and are provided without any warranties, specific or implied. Therefore, the analysis results provided by ClinGenics constitute data which will require further assessment and interpretation by a skilled genetics professional, physician or medical doctor. The variant interpretation services are provided for consultation and advisory purposes only and should not be viewed as a substitute for medical advice.
ClinGenics will make every effort to provide state-of-the-art variant interpretation analysis. However, you hereby accept that the results of these analyses rely also on data and information contained in numerous external databases and in scientific publications available at the time, the contents of which may be incomplete and/or inaccurate and may therefore affect the results of variant interpretation provided by ClinGenics. No warranty is provided in this respect, in particular that such data or information is accurate.
The limitations of the services provided by the ClinGenics EMA® – Exome Management Application pipeline decision-support software include, but are not limited to:
- The user-provided sample information, as stated in the Sample Submission Form (clinical and phenotype data, etc), may be inaccurate or misleading.
- Poor NGS data quality, as reflected in the submitted vcf file. Interpretation of indels may be less accurate than the interpretation of single base substitutions (SNPs) due to potential NGS sequencing errors.
- False positive (artifactual variants) and false negative (missed variants) in the sequencing or base calling stages are reflected in the submitted vcf file and will affect interpretation results.
- There is no standard for exome clinical analysis, and the use of different methods (mapping algorithms, base calling and annotation tools) may also influence the final interpretation.
- Variants in genomic regions outside the defined exome enrichment list are not included in the analysis.
- Gene and/or exon coverage may not be uniform, resulting in regions with low or no sequence coverage.
- Structural variations, large deletions or insertions, short tandem repeats (STR’s) expansions and nucleotide repeat expansions are not evaluated and not reported.
- Clinically significant variants must be validated by confirmatory Sanger sequencing prior to final clinical assessment.
- Incidental findings in genes recommended by the ACMG are not evaluated as this is a RESEARCH USE ONLY service.
- Interpretation of the results should be finally reviewed by the referring/treating physician, who is ultimately responsible for providing a final clinical diagnosis.
- Genetic counseling is strongly recommended to explain reported results to the tested individuals.
- The ClinGenics Exome Management Application–EMA® pipeline software by design will currently only prioritize, evaluate and report variants in known disease-associated genes and is not by itself a tool for discovering novel disease genes.
Disclaimer – Legal Notice
ClinGenics Ltd. will not be liable for any modification or suspension of its services or for any loss of content or data provided by you.
ClinGenics does not require, collect or store personally identifiable information associated with the submitted data files. ClinGenics will not accept and process submitted data files which may contain patient-specific identifiers (e.g. names, address, etc.).
ClinGenics will not share any information or data with any other person or company.
- Harrison SM, Riggs ER, Maglott DR et al Using ClinVar as a resource to support variant interpretation. Curr. Protoc. Hum. Genet. 2016;89; 8.16.1-8.16.23.
- Köhler S, Vasilevsky NA, Engelstad M et al The Human Phenotype Ontology in 2017. Nucleic Acids Res. 2017;45:D865-D876.
- Landrum, M. J. et al ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 2016;44:D862-D868.
- Lek M, Karczewski KJ, Minikel EV et al Analysis of protein-coding genetic variation in 60,706 humans. Nature 2016;536:285–291.
- Liu X, Jian X, and Boerwinkle E. dbNSFP: a lightweight database of human non-synonymous SNPs and their functional predictions. Human Mutation 2011;32:894-899.
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- Liu X, Wu C, Li C and Boerwinkle E. dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Non-synonymous and Splice Site SNVs. Human Mutation 2016;37:235-241.
- Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD). World Wide Web URL: https://omim.org/
- Rehm HL, Berg JS, Brooks LD et al ClinGen-the Clinical Genome Resource. N. Engl. J. Med. 2015;372:2235-2242.
- The 1000 Genomes Project Consortium, Auton A, Brooks LD et al A global reference for human genetic variation. Nature 2015;526:68-74.
and many more…
- Konialis C, Hagnefelt B, Karapanou S, Pangalos C. Whole exome sequencing in a newborn with severe distal arthrogryposis reveals homozygosity for a paternal ECEL1 gene mutation as a result of uniparental paternal isodisomy for chromosome 2 (manuscript in preparation)
- Konialis C, Lilakos K, Hagnefelt B, Karapanou S, Pangalos C. A Microdeletion at Xp11.22 Detected by Whole Exome Sequencing Confirms SHROOM4 Association with Stocco dos Santos Syndrome and XLID in a Large Kindred (manuscript submitted)
- Gontika M, Konialis C, Pangalos C, Papavasiliou A. Novel SCN1A and GABRA1 gene mutations with diverse phenotypic features and the question on the existence of a broader spectrum of Dravet Syndrome Child Neurol Open. 2017 May 8;4:2329048X17706794
- Konialis C, Asimakopoulos E, Hagnefelt B, Karapanou S, Sotiriadis A, Pangalos C. Prenatal diagnosis of X-linked Myopathy associated with a VMA21 gene mutation afforded through a novel targeted exome sequencing strategy applied in fetuses with abnormal ultrasound findings. Clin Case Rep 2017;5:308–311.
- Pangalos C, Hagnefelt H, Lilakos K, Konialis C. First applications of a targeted exome sequencing approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with associated gene defects. Peer J 2016 Apr 26;4:e1955.