Comparison of Exome and Genome Sequencing Technologies for the Complete Capture of Protein‐Coding Regions

For next‐generation sequencing technologies, sufficient base‐pair coverage is the foremost requirement for the reliable detection of genomic variants. We investigated whether whole‐genome sequencing (WGS) platforms offer improved coverage of coding regions compared with whole‐exome sequencing (WES) platforms, and compared single‐base coverage for a large set of exome and genome samples. We find that WES platforms have improved considerably in the last years, but at comparable sequencing depth, WGS outperforms WES in terms of covered coding regions. At higher sequencing depth (95x–160x), WES successfully captures 95% of the coding regions with a minimal coverage of 20x, compared with 98% for WGS at 87‐fold coverage. Three different assessments of sequence coverage bias showed consistent biases for WES but not for WGS. We found no clear differences for the technologies concerning their ability to achieve complete coverage of 2,759 clinically relevant genes. We show that WES performs comparable to WGS in terms of covered bases if sequenced at two to three times higher coverage. This does, however, go at the cost of substantially more sequencing biases in WES approaches. Our findings will guide laboratories to make an informed decision on which sequencing platform and coverage to choose.     

An Evaluation of Copy Number Variation Detection Tools from Whole‐Exome Sequencing Data

Copy number variation (CNV) has been found to play an important role in human disease. Next‐generation sequencing technology, including whole‐genome sequencing (WGS) and whole‐exome sequencing (WES), has become a primary strategy for studying the genetic basis of human disease. Several CNV calling tools have recently been developed on the basis of WES data. However, the comparative performance of these tools using real data remains unclear. An objective evaluation study of these tools in practical research situations would be beneficial. Here, we evaluated four well‐known WES‐based CNV detection tools (XHMM, CoNIFER, ExomeDepth, and CONTRA) using real data generated in house. After evaluation using six metrics, we found that the sensitive and accurate detection of CNVs in WES data remains challenging despite the many algorithms available. Each algorithm has its own strengths and weaknesses. None of the exome‐based CNV calling methods performed well in all situations; in particular, compared with CNVs identified from high coverage WGS data from the same samples, all tools suffered from limited power. Our evaluation provides a comprehensive and objective comparison of several well‐known detection tools designed for WES data, which will assist researchers in choosing the most suitable tools for their research needs.     

基于隐马尔可夫模型的全外显子测序拷贝数变异检测算法研究

拷贝数变异(CNV)是基因组变异的一种非平衡结构变异,与癌症等许多复杂疾病相关.目前,基于隐马尔可夫模型(HMM)的CNV检测算法已经成为基因检测研究中的一大热点,但是对于这些基于HMM的CNV检测工具并没有进行过系统的比较,导致在应用时选择困难.选取5种具有代表性的基于HMM的CNV检测工具:ExomeDepth、E...     

Genetic Analysis of Korean Adult Patients with Nontuberculous Mycobacteria Suspected of Primary Ciliary Dyskinesia Using Whole Exome Sequencing

PurposeNontuberculous mycobacteria (NTM) is ubiquitous in the environment, but NTM lung disease (NTM-LD) is uncommon. Since exposure to NTM is inevitable, patients who develop NTM-LD are likely to have specific susceptibility factors, such as primary ciliary dyskinesia (PCD). PCD is a genetically heterogeneous disorder of motile cilia and is characterized by chronic respiratory tract infection, organ laterality defect, and infertility. In this study, we performed whole exome sequencing (WES) and investigated the genetic characteristics of adult NTM patients with suspected PCD.Materials and MethodsWES was performed in 13 NTM-LD patients who were suspected of having PCD by clinical symptoms and/or ultrastructural ciliary defect observed by transmission electron microscopy. A total of 45 PCD-causing genes, 23 PCD-candidate genes, and 990 ciliome genes were analyzed.ResultsFour patients were found to have biallelic loss-of-function (LoF) variants in the following PCD-causing genes: CCDC114, DNAH5, HYDIN, and NME5. In four other patients, only one LoF variant was identified, while the remaining five patients did not have any LoF variants.ConclusionAt least 30.8% of NTM-LD patients who were suspected of having PCD had biallelic LoF variants, and an additional 30.8% of patients had one LoF variant. Therefore, PCD should be considered in patients with NTM-LD with symptoms or signs suspicious of PCD.     

Genome sequencing of mucosal melanomas reveals that they are driven by distinct mechanisms from cutaneous melanoma

Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun‐exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light‐ or tobacco smoke‐induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than in cutaneous melanomagenesis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Detection of Clinically Relevant Copy Number Variants with Whole‐Exome Sequencing

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders. This has resulted in the widespread application of genomic microarrays as a first‐tier diagnostic tool for CNV detection. More recently, whole‐exome sequencing (WES) has been proven successful for the detection of clinically relevant point mutations and small insertion–deletions exome wide. We evaluate the utility of short‐read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compare these results to data from two independent high‐resolution microarrays. Eleven of the 12 clinically relevant CNVs were detected via read‐depth analysis of WES data; a heterozygous single‐exon deletion remained undetected by all algorithms evaluated. Although the detection power of WES for small CNVs currently does not match that of high‐resolution microarray platforms, we show that the majority (88%) of rare coding CNVs containing three or more exons are successfully identified by WES. These results show that the CNV detection resolution of WES is comparable to that of medium‐resolution genomic microarrays commonly used as clinical assays. The combined detection of point mutations, indels, and CNVs makes WES a very attractive first‐tier diagnostic test for genetically heterogeneous disorders.     

A Post‐Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.     

Genomics really gets personal: how exome and whole genome sequencing challenge the ethical framework of human genetics research

Exome sequencing (ES) and whole genome sequencing (WGS) putatively identify all adverse functional alleles of protein-coding genes. Accordingly, while ES/WGS are transformative new tools for gene discovery in human and medical genetics research, they also generate new manifestations of ethical issues related to the consent process, data sharing, and return of results. These manifestations have yet to be comprehensively framed, due in part to the rapidity with which new technologies for ES/WGS are being applied and because of a lack of empirical data to provide guidance. Accordingly, researchers, funding agencies, and policy makers have largely dealt with these issues intuitively. We explain how use of ES/WGS challenges: (i) models under which informed consent is typically obtained; (ii) how harms associated with data sharing are considered; and (iii) the nature of obligations surrounding unanticipated findings. We provide broad guidance about interim ways to contend with these issues and make broad recommendations for areas for novel resource and policy development.     

Informing Cancer Prevention Strategies for African Americans: The Relationship of African American Acculturation to Fruit,Vegetable, and Fat Intake

Acculturation has been associated with health-related behaviors in African Americans. We sought to determine if there is a relationship between acculturation and dietary intake in African Americans. African Americans in the PREMIER trial completed the African American Acculturation Scale (AAAS) and 2 nonconsecutive 24-h dietary recalls (n = 238). Analysis of variance (ANOVA) and canonical correlation were used to assess relationships between acculturation and dietary intakes. Canonical correlation (p = 0.05) showed that traditional African Americans had lower intakes of fruits/vegetables and milk/dairy with higher intakes of fats, meat, and nuts. This pattern was supported by differences in the ANOVA. African American acculturation is related to dietary intake. These findings have implications for the design of cancer-related public health messages targeted to African Amercans.     

Informed and patient-centered decision-making in the primary care visits of African Americans with depression

Objective

We examined the prevalence and extent of informed decision-making (IDM) and patient-centered decision-making (PCDM) in primary care visits of African Americans with depression.

Revision and validation of the medication adherence self-efficacy scale (MASES) in hypertensive African Americans

Study purpose was to revise and examine the validity of the Medication Adherence Self-Efficacy Scale (MASES) in an independent sample of 168 hypertensive African Americans: mean age 54 years (SD = 12.36); 86% female; 76% high school education or greater. Participants provided demographic information; completed the MASES, self-report and electronic measures of medication adherence at baseline and three months. Confirmatory (CFA), exploratory (EFA) factor analyses, and classical test theory (CTT) analyses suggested that MASES is unidimensional and internally reliable. Item response theory (IRT) analyses led to a revised 13-item version of the scale: MASES-R. EFA, CTT, and IRT results provide a foundation of support for MASES-R reliability and validity for African Americans with hypertension. Research examining MASES-R psychometric properties in other ethnic groups will improve generalizability of findings and utility of the scale across groups. The MASES-R is brief, quick to administer, and can capture useful data on adherence self-efficacy.     

A Robust Approach for Blind Detection of Balanced Chromosomal Rearrangements with Whole‐Genome Low‐Coverage Sequencing

Balanced chromosomal rearrangement (or balanced chromosome abnormality, BCA) is a common chromosomal structural variation. Next‐generation sequencing has been reported to detect BCA‐associated breakpoints with the aid of karyotyping. However, the complications associated with this approach and the requirement for cytogenetics information has limited its application. Here, we provide a whole‐genome low‐coverage sequencing approach to detect BCA events independent of knowing the affected regions and with low false positives. First, six samples containing BCAs were used to establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out the false‐positive results with a control cohort and the concomitant mapping information, we could directly detect BCA events for each sample. Through optimizing the read depth, BCAs in all samples could be blindly detected with only 120 million read pairs per sample for data from a small‐insert library and 30 million per sample for data from nonsize‐selected mate‐pair library. This approach was further validated using another 13 samples that contained BCAs. Our approach advances the application of high‐throughput whole‐genome low‐coverage analysis for robust BCA detection—especially for clinical samples—without the need for karyotyping.     

Perspectives of clinical genetics professionals toward genome sequencing and incidental findings: a survey study

The introduction of clinical genome‐wide sequencing raises complex issues regarding the management of incidental findings. However, there is a lack of empirical studies assessing views of providers involved in potential disclosure of such findings. In an anonymous survey of 279 clinical genetics professionals, we found that the vast majority of participants agreed that they were interested in knowing about clinically actionable incidental findings in themselves (96%) and their child (99%), and they reported that these types of findings should be disclosed in adult (96%) and minor (98%) patients. Approximately three‐fourths agreed that they were personally interested in knowing about an adult‐onset clinically actionable disease (78%) and a childhood‐onset non‐clinically actionable disease (75%) in their child. A similar percentage of participants (70%) felt that these two types of findings should be disclosed to patients. Forty‐four percent of participants wanted to know about an incidental finding that indicates an adult‐onset non‐clinically actionable condition in themselves and 31% wanted to know about this type of information in their child. Findings from this study revealed participants' views highly dependent on clinical actionability. Further research is needed with a broader population of geneticists to increase generalizability, and with diverse patients to assess their perspectives about results disclosure from clinical sequencing.     

Whole exome sequencing in a patient with uniparental disomy of chromosome 2 and a complex phenotype

Whole exome sequencing and chromosomal microarrays are two powerful technologies that have transformed the ability of researchers to search for potentially causal variants in human disease. This study combines these tools to search for causal variants in a patient found to have maternal uniparental isodisomy of chromosome 2. This subject has a complex phenotype including skeletal and renal dysplasia, immune deficiencies, growth failure, retinal degeneration and ovarian insufficiency. Eighteen non‐synonymous, rare homozygous variants were identified on chromosome 2. Additionally, five genes with compound heterozygous mutations were detected on other chromosomes that could lead to a disease phenotype independent of the uniparental disomy found in this case. Several candidate genes with potential connection to the phenotype are described but none are definitively proven to be causal. This study highlights the potential for detection of a large number of candidate genes using whole exome sequencing complicating interpretation in both the research and clinical settings. Forums must be created for publication and sharing of detailed phenotypic and genotypic reports to facilitate further biological discoveries and clinical counseling.