Comparison of Mendeliome exome capture kits for use in clinical diagnostics.

Next generation sequencing has disrupted genetic testing, allowing far more scope in the tests applied. The appropriate sections of the genome to be tested can now be readily selected, from single mutations to whole-genome sequencing. One product offering within this spectrum are focused exomes, targeting ~5,000 genes know to be implicated in human disease. These are designed to offer a flexible platform offering high diagnostic yield with a reduction in sequencing requirement compared to whole exome sequencing. Here, we have undertaken sequencing of control DNA samples and compare two kits, the Illumina TruSight One and the Agilent SureSelect Focused Exome. Characteristics of the kits are comprehensively evaluated. Despite the larger design region of the Agilent kit, we find that the Illumina kit performs better in terms of gene coverage, as well as coverage of clinically relevant loci. We provide exhaustive coverage statistics for each kit to aid the assessment of their suitability and provide read data for control DNA samples to allow for bioinformatic benchmarking by users developing pipelines for these data.

A small gene sequencing panel realises a high diagnostic rate in patients with congenital nystagmus following basic phenotyping.

Nystagmus is a disorder of uncontrolled eye movement and can occur as an isolated trait (idiopathic INS, IINS) or as part of multisystem disorders such as albinism, significant visual disorders or neurological disease. Eighty-one unrelated patients with nystagmus underwent routine ocular phenotyping using commonly available phenotyping methods and were grouped into four sub-cohorts according to the level of phenotyping information gained and their findings. DNA was extracted and sequenced using a broad utility next generation sequencing (NGS) gene panel. A clinical subpanel of genes for nystagmus/albinism was utilised and likely causal variants were prioritised according to methods currently employed by clinical diagnostic laboratories. We determine the likely underlying genetic cause for 43.2% of participants with similar yields regardless of prior phenotyping. This study demonstrates that a diagnostic workflow combining basic ocular phenotyping and a clinically available targeted NGS panel, can provide a high diagnostic yield for patients with infantile nystagmus, enabling access to disease specific management at a young age and reducing the need for multiple costly, often invasive tests. By describing diagnostic yield for groups of patients with incomplete phenotyping data, it also permits the subsequent design of 'real-world' diagnostic workflows and illustrates the changing role of genetic testing in modern diagnostic workflows for heterogeneous ophthalmic disorders.

Autoimmunity/inflammation in a monogenic primary immunodeficiency cohort.

Primary immunodeficiencies (PIDs) are rare inborn errors of immunity that have a heterogeneous phenotype that can include severe susceptibility to life-threatening infections from multiple pathogens, unique sensitivity to a single pathogen, autoimmune/inflammatory (AI/I) disease, allergies and/or malignancy. We present a diverse cohort of monogenic PID patients with and without AI/I diseases who underwent clinical, genetic and immunological phenotyping. Novel pathogenic variants were identified in IKBKG, CTLA4, NFKB1, GATA2, CD40LG and TAZ as well as previously reported pathogenic variants in STAT3, PIK3CD, STAT1, NFKB2 and STXBP2. AI/I manifestations were frequently encountered in PIDs, including at presentation. Autoimmunity/inflammation was multisystem in those effected, and regulatory T cell (Treg) percentages were significantly decreased compared with those without AI/I manifestations. Prednisolone was used as the first-line immunosuppressive agent in all cases, however steroid monotherapy failed long-term control of autoimmunity/inflammation in the majority of cases and additional immunosuppression was required. Patients with multisystem autoimmunity/inflammation should be investigated for an underlying PID, and in those with PID early assessment of Tregs may help to assess the risk of autoimmunity/inflammation.

Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B).

Oculocutaneous albinism (OCA) and ocular albinism (OA) are inherited disorders of melanin biosynthesis, resulting in loss of pigment and severe visual deficits. OCA encompasses a range of subtypes with overlapping, often hypomorphic phenotypes. OCA1 is the most common cause of albinism in European populations and is inherited through autosomal recessive mutations in the Tyrosinase (TYR) gene. However, there is a high level of reported missing heritability, where only a single heterozygous mutation is found in TYR. This is also the case for other OCA subtypes including OCA2 caused by mutations in the OCA2 gene. Here we have interrogated the genetic cause of albinism in a well phenotyped, hypomorphic albinism population by sequencing a broad gene panel and performing segregation studies on phenotyped family members. Of eighteen probands we can confidently diagnose three with OA and OCA2, and one with a PAX6 mutation. Of six probands with only a single heterozygous mutation in TYR, all were found to have the two common variants S192Y and R402Q. Our results suggest that a combination of R402Q and S192Y with a deleterious mutation in a 'tri-allelic genotype' can account for missing heritability in some hypomorphic OCA1 albinism phenotypes.

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Erratum to: a SNP profiling panel for sample tracking in whole-exome sequencing studies.

This is an Erratum to Genome Medicine 2013, 5:89, highlighting an error in Table 1 of the original article. Please see related article: http://genomemedicine.com/content/5/9/89.[This corrects the article DOI: 10.1186/gm492.].

A SNP profiling panel for sample tracking in whole-exome sequencing studies.

Whole-exome sequencing provides a cost-effective means to sequence protein coding regions within the genome, which are significantly enriched for etiological variants. We describe a panel of single nucleotide polymorphisms (SNPs) to facilitate the validation of data provenance in whole-exome sequencing studies. This is particularly significant where multiple processing steps necessitate transfer of sample custody between clinical, laboratory and bioinformatics facilities. SNPs captured by all commonly used exome enrichment kits were identified, and filtered for possible confounding properties. The optimised panel provides a simple, yet powerful, method for the assignment of intrinsic, highly discriminatory identifiers to genetic samples.

Review of massively parallel DNA sequencing technologies.

Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.

A standardized framework for the validation and verification of clinical molecular genetic tests.

The validation and verification of laboratory methods and procedures before their use in clinical testing is essential for providing a safe and useful service to clinicians and patients. This paper outlines the principles of validation and verification in the context of clinical human molecular genetic testing. We describe implementation processes, types of tests and their key validation components, and suggest some relevant statistical approaches that can be used by individual laboratories to ensure that tests are conducted to defined standards.

Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning.

BACKGROUND: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning. METHODS: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing. RESULTS: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%-93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%-99.4% and 93.1%-95.5%, respectively. CONCLUSIONS: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.

Semi-automated unidirectional sequence analysis for mutation detection in a clinical diagnostic setting.

BACKGROUND: The past 10 years have seen an improvement in sequence data quality due to the introduction of capillary sequencers and new sequencing chemistries. In parallel, new software programs for automated mutation detection have been developed. We evaluated the sensitivity of semiautomated unidirectional sequence analysis for the detection of heterozygous base substitutions using the Mutation Surveyor software package. METHODS: Detection rates for heterozygous base substitutions in 29 genes by automated and visual inspection were compared. Examples of heterozygous bases not detected in one direction during bidirectional analysis were also sought through a national survey of United Kingdom (UK) genetics laboratories. Sequence quality was assessed in a consecutive cohort of 50 patients for whom the 39 exons of the ABCC8 gene had been sequenced in one direction. RESULTS: A total of 701 different heterozygous base substitutions were detected by the software with no false negatives (sensitivity >or=99.57%). Four examples of heterozygous bases missed in one direction during bidirectional analysis were reported. Two were detected using unidirectional analysis settings, and the other two bases had low-quality scores. Of the 1950 amplicons examined, 97.2% had a quality score >or=30 and an average PHRED-like score >or=50 for the defined region of interest, and 98.1% of the 323,650 bases had a PHRED score >40. CONCLUSIONS: We found no evidence to support a requirement for bidirectional sequencing. Semiautomated analysis of good quality unidirectional sequence data has high sensitivity and is suitable for heterozygote mutation scanning in clinical diagnostic laboratories. Further work is required to determine minimum quality parameters for semiautomated analysis.