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Front Genet. 2014 Feb 12;5:16. doi: 10.3389/fgene.2014.00016. eCollection 2014.

The struggle to find reliable results in exome sequencing data: filtering out Mendelian errors.

Author information

1
Division of Rheumatology, Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA ; Medical Scientist Training Program, University of Cincinnati College of Medicine, Cincinnati OH, USA.
2
Division of Rheumatology, Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA ; Department of Veterans Affairs, Veterans Affairs Medical Center - Cincinnati, Cincinnati OH, USA.
3
Genomic Medicine Institute, Geisinger Health System, Danville PA, USA.
4
Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA.
5
Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA.
6
Division of Infectious Disease, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA.

Abstract

Next Generation Sequencing studies generate a large quantity of genetic data in a relatively cost and time efficient manner and provide an unprecedented opportunity to identify candidate causative variants that lead to disease phenotypes. A challenge to these studies is the generation of sequencing artifacts by current technologies. To identify and characterize the properties that distinguish false positive variants from true variants, we sequenced a child and both parents (one trio) using DNA isolated from three sources (blood, buccal cells, and saliva). The trio strategy allowed us to identify variants in the proband that could not have been inherited from the parents (Mendelian errors) and would most likely indicate sequencing artifacts. Quality control measurements were examined and three measurements were found to identify the greatest number of Mendelian errors. These included read depth, genotype quality score, and alternate allele ratio. Filtering the variants on these measurements removed ~95% of the Mendelian errors while retaining 80% of the called variants. These filters were applied independently. After filtering, the concordance between identical samples isolated from different sources was 99.99% as compared to 87% before filtering. This high concordance suggests that different sources of DNA can be used in trio studies without affecting the ability to identify causative polymorphisms. To facilitate analysis of next generation sequencing data, we developed the Cincinnati Analytical Suite for Sequencing Informatics (CASSI) to store sequencing files, metadata (eg. relatedness information), file versioning, data filtering, variant annotation, and identify candidate causative polymorphisms that follow either de novo, rare recessive homozygous or compound heterozygous inheritance models. We conclude the data cleaning process improves the signal to noise ratio in terms of variants and facilitates the identification of candidate disease causative polymorphisms.

KEYWORDS:

CASSI; Mendel errors; Mendelian errors; disease causative polymorphisms; next-generation sequencing; variant filtering; whole exome sequencing

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