Regulating whole exome sequencing as a diagnostic test

In the last decade, there has been a flood of new technology in the sequencing arena. The onset of next-generation sequencing (NGS) technology has resulted in the vast increase in genetic diagnostic testing available to the ordering physician. Whole exome sequencing (WES) has become available as a diagnostic test performed in certified clinical laboratories. This has led to increased presence in the diagnostic marketplace, increased consumer awareness, and the question has been raised by various stakeholders to whether there is sufficient stringent regulation of WES and other NGS-based tests. We discuss the various WES services currently available in the marketplace, current regulation of WES as a laboratory developed test, the proposed FDA involvement in its oversight as well as the response of various laboratory groups that provide these diagnostic services. Overall, a rigorous process oversight and assessment of inter-lab reproducibility is strongly warranted for WES as it is used as a diagnostic test, but regulation should be mindful of the excessive administrative burden on academic and smaller diagnostic laboratories.     

Rapid gene identification in a Chinese osteopetrosis family by whole exome sequencing

Osteopetrosis is a rare genetically heterogeneous disorder of bone metabolism characterized by increased skeleton density. In the past, standard methods for genetic diagnosis of osteopetrosis have primarily been performed by candidate gene screening and positional cloning. However, these methods are time and labor consumptive; and the genetic basis of approximately 30% of the cases is yet to be elucidated. Here, we employed whole exome sequencing of two affected individuals from an osteopetrosis family to identify a candidate mutation in CLCN7 (Y99C). It was identified from a total of 1757 and 1728 genetic variations found in either patient, which were then distilled using filtering strategies and confirmed using Sanger sequencing. We identified this mutation in six family members, while not in population matched controls. This mutation was previously found in osteopetrosis patients by other researchers. Our evolutionary analysis also indicated that it is under extremely high selective pressure, and is likely to be critical for the correct function of ClC-7, and thus is likely to be the responsible cause of disease. Collectively, our data further indicated that mutation (Y99C) may be a cause of osteopetrosis, and highlights the use of whole exome sequencing as a valuable approach to identifying disease mutations in a cost and time efficient manner.     

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.     

Challenges in whole exome sequencing: an example from hereditary deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ～6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease.     

Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing

Genomic sequence comparisons between individuals are usually restricted to the analysis of single nucleotide polymorphisms (SNPs). While the interrogation of SNPs is efficient, they are not the only form of divergence between genomes. In this report, we expand the scope of polymorphism detection by investigating the occurrence of double nucleotide polymorphisms (DNPs) and triple nucleotide polymorphisms (TNPs), in which two or three consecutive nucleotides are altered compared to the reference sequence. We have found such DNPs and TNPs throughout two complete genomes and eight exomes. Within exons, these novel polymorphisms are over-represented amongst protein-altering variants; nearly all DNPs and TNPs result in a change in amino acid sequence and, in some cases, two adjacent amino acids are changed. DNPs and TNPs represent a potentially important new source of genetic variation which may underlie human disease and they should be included in future medical genetics studies. As a confirmation of the damaging nature of xNPs, we have identified changes in the exome of a glioblastoma cell line that are important in glioblastoma pathogenesis. We have found a TNP causing a single amino acid change in LAMC2 and a TNP causing a truncation of HUWE1.     

Variant detection sensitivity and biases in whole genome and exome sequencing

Background

Less than two percent of the human genome is protein coding, yet that small fraction harbours the majority of known disease causing mutations. Despite rapidly falling whole genome sequencing (WGS) costs, much research and increasingly the clinical use of sequence data is likely to remain focused on the protein coding exome. We set out to quantify and understand how WGS compares with the targeted capture and sequencing of the exome (exome-seq), for the specific purpose of identifying single nucleotide polymorphisms (SNPs) in exome targeted regions.

Results

We have compared polymorphism detection sensitivity and systematic biases using a set of tissue samples that have been subject to both deep exome and whole genome sequencing. The scoring of detection sensitivity was based on sequence down sampling and reference to a set of gold-standard SNP calls for each sample. Despite evidence of incremental improvements in exome capture technology over time, whole genome sequencing has greater uniformity of sequence read coverage and reduced biases in the detection of non-reference alleles than exome-seq. Exome-seq achieves 95% SNP detection sensitivity at a mean on-target depth of 40 reads, whereas WGS only requires a mean of 14 reads. Known disease causing mutations are not biased towards easy or hard to sequence areas of the genome for either exome-seq or WGS.

Conclusions

From an economic perspective, WGS is at parity with exome-seq for variant detection in the targeted coding regions. WGS offers benefits in uniformity of read coverage and more balanced allele ratio calls, both of which can in most cases be offset by deeper exome-seq, with the caveat that some exome-seq targets will never achieve sufficient mapped read depth for variant detection due to technical difficulties or probe failures. As WGS is intrinsically richer data that can provide insight into polymorphisms outside coding regions and reveal genomic rearrangements, it is likely to progressively replace exome-seq for many applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-247) contains supplementary material, which is available to authorized users.     

Screening the human exome: a comparison of whole genome and whole transcriptome sequencing

Background  

There is considerable interest in the development of methods to efficiently identify all coding variants present in large sample sets of humans. There are three approaches possible: whole-genome sequencing, whole-exome sequencing using exon capture methods, and RNA-Seq. While whole-genome sequencing is the most complete, it remains sufficiently expensive that cost effective alternatives are important.     

An integrative variant analysis suite for whole exome next-generation sequencing data

Background  

Whole exome capture sequencing allows researchers to cost-effectively sequence the coding regions of the genome. Although the exome capture sequencing methods have become routine and well established, there is currently a lack of tools specialized for variant calling in this type of data.     

CANOES: detecting rare copy number variants from whole exome sequencing data

We present CANOES, an algorithm for the detection of rare copy number variants from exome sequencing data. CANOES models read counts using a negative binomial distribution and estimates variance of the read counts using a regression-based approach based on selected reference samples in a given dataset. We test CANOES on a family-based exome sequencing dataset, and show that its sensitivity and specificity is comparable to that of XHMM. Moreover, the method is complementary to Gaussian approximation-based methods (e.g. XHMM or CoNIFER). When CANOES is used in combination with these methods, it will be possible to produce high accuracy calls, as demonstrated by a much reduced and more realistic de novo rate in results from trio data.     

Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

Owing to rapid advancements in NGS (next generation sequen-cing), genomic alteration is now considered an essential pre-dictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was con-sidered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly com-pared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture re-gion, which might lead to different values of TMB; the evalu-ation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evalu-ated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R² = 0.887). This was also reflected in clinical samples (n = 100), which showed R² of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings.     

Identification of novel candidate genes in heterotaxy syndrome patients with congenital heart diseases by whole exome sequencing

Heterotaxy syndrome (HS) involves dysfunction of multiple systems resulting from abnormal left-right (LR) body patterning. Most HS patients present with complex congenital heart diseases (CHD), the disability and mortality of HS patients are extremely high. HS has great heterogeneity in phenotypes and genotypes, which have rendered gene discovery challenging. The aim of this study was to identify novel genes that underlie pathogenesis of HS patients with CHD. Whole exome sequencing was performed in 25 unrelated HS cases and 100 healthy controls; 19 nonsynonymous variants in 6 novel candidate genes (FLNA, ITGA1, PCNT, KIF7, GLI1, KMT2D) were identified. The functions of candidate genes were further analyzed in zebrafish model by CRISPR/Cas9 technique. Genome-editing was successfully introduced into the gene loci of flna, kmt2d and kif7, but the phenotypes were heterogenous. Disruption of each gene disturbed normal cardiac looping while kif7 knockout had a more prominent effect on liver budding and pitx2 expression. Our results revealed three potential HS pathogenic genes with probably different molecular mechanisms.     

Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome

Background

Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.

Methodology/Principal Findings

We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient''s mother, father and young brother, all of whom are normoglycemic.

Conclusions/Significance

WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.     

A new tool for prioritization of sequence variants from whole exome sequencing data

Background

Whole exome sequencing (WES) has provided a means for researchers to gain access to a highly enriched subset of the human genome in which to search for variants that are likely to be pathogenic and possibly provide important insights into disease mechanisms. In developing countries, bioinformatics capacity and expertise is severely limited and wet bench scientists are required to take on the challenging task of understanding and implementing the barrage of bioinformatics tools that are available to them.

Results

We designed a novel method for the filtration of WES data called TAPER? (Tool for Automated selection and Prioritization for Efficient Retrieval of sequence variants).

Conclusions

TAPER? implements a set of logical steps by which to prioritize candidate variants that could be associated with disease and this is aimed for implementation in biomedical laboratories with limited bioinformatics capacity. TAPER? is free, can be setup on a Windows operating system (from Windows 7 and above) and does not require any programming knowledge. In summary, we have developed a freely available tool that simplifies variant prioritization from WES data in order to facilitate discovery of disease-causing genes.

     

High-performance single-chip exon capture allows accurate whole exome sequencing using the Illumina Genome Analyzer

Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for whole exome sequencing using the Illumina Genome Analyzer (GA) platform. The protocol involves two-stage library construction. The specificity of exome enrichment was approximately 80% with 95.6% even coverage of the 34 Mb target region at an average sequencing depth of 33-fold. Comparison of our results with whole genome shot-gun resequencing results showed that the exome SNP calls gave only 0.97% false positive and 6.27% false negative variants. Our protocol is also well suited for use with whole genome amplified DNA. The results presented here indicate that there is a promising future for large-scale population genomics and medical studies using a whole exome sequencing approach.     

CODEX: a normalization and copy number variation detection method for whole exome sequencing

High-throughput sequencing of DNA coding regions has become a common way of assaying genomic variation in the study of human diseases. Copy number variation (CNV) is an important type of genomic variation, but detecting and characterizing CNV from exome sequencing is challenging due to the high level of biases and artifacts. We propose CODEX, a normalization and CNV calling procedure for whole exome sequencing data. The Poisson latent factor model in CODEX includes terms that specifically remove biases due to GC content, exon capture and amplification efficiency, and latent systemic artifacts. CODEX also includes a Poisson likelihood-based recursive segmentation procedure that explicitly models the count-based exome sequencing data. CODEX is compared to existing methods on a population analysis of HapMap samples from the 1000 Genomes Project, and shown to be more accurate on three microarray-based validation data sets. We further evaluate performance on 222 neuroblastoma samples with matched normals and focus on a well-studied rare somatic CNV within the ATRX gene. We show that the cross-sample normalization procedure of CODEX removes more noise than normalizing the tumor against the matched normal and that the segmentation procedure performs well in detecting CNVs with nested structures.     

一站式全基因组和外显子组测序数据自动分析软件(SeqMule)

SeqMule可根据调用的人类基因组和外显子组数据自动调节变量,对所有测序数据的单核苷酸多态性(Single nucleotide polymorphism,SNP)进行分析和注释。目的:通过对两名痛风患者的实验数据进行分析,详细地为生物信息学研究人员介绍了SeqMule软件,以期为全基因组和外显子组测序数据提供一站式的分析途径。方法:基于SeqMule内置的BWA(BurrowsWheeler Aligner)、GATK(The Genome Analysis Toolkit)、SAMtools、Freebayes比对和分析工具,以两名痛风患者的DNA测序数据分析为例,本文详细地论述了SeqMule的特点及操作,并对两名患者的外显子测序数据进行了自动化比对与SNP分析。发现SeqMule优化了很多分析软件存在的一些问题,可以对外显子组和全基因组测序数据实现全面、灵活、高效地自动化分析,能更好地分析高通量测序数据,最终提升数据分析的一致性和准确性。     

Analysis of the inheritance pattern of a Chinese family with phaeochromocytomas through whole exome sequencing

Phaeochromocytomas (PCCs) and paragangliomas (PGLs) are rare, catecholamine-producing tumors. Most familial PCC/PGLs have been detected to be autosomal dominantly inherited. However, this study was undertaken in a family with PCCs to determine candidate genes in a dominant or recessive inheritance pattern. After excluding mutations in ten PCC/PGL susceptibility genes by Sanger sequencing, we used whole exome sequencing for screening on the four family members to discover novel candidate genes associated with PCCs. Based on the inexistence of non-synonymous mutations or indels in the ten known genes and the structure of this pedigree, 3 damaging loci with dominant inheritance pattern, and 5 damaging loci with recessive homozygous inheritance pattern and 6 damaging genes with compound heterozygous inheritance pattern were narrowed down to indicate the association with PCCs. According to the Gene Ontology (GO) category analysis on the combined results, cell adhesion showed the most significant enrichment.     

The role of phenotype in gene discovery in the whole genome sequencing era

As whole genome sequence becomes a routine component of gene discovery studies in humans, we will have an exhaustive catalog of genetic variation and the challenge becomes understanding the phenotypic consequences of these variants. Statistical genetic methods and analytical approaches that are concerned with optimizing phenotypes for gene discovery for complex traits offer two general categories of advantages. They may increase power to localize genes of interest and also aid in interpreting associations between genetic variants and disease outcomes by suggesting potential mechanisms and pathways through which genes may affect outcomes. Such phenotype optimization approaches include use of allied phenotypes such as symptoms or ages of onset to reduce genetic heterogeneity within a set of cases, study of quantitative risk factors or endophenotypes, joint analyses of related phenotypes, and derivation of new phenotypes designed to extract independent measures underlying the correlations among a set of related phenotypes through approaches such as principal components. New opportunities are also presented by technological advances that permit efficient collection of hundreds or thousands of phenotypes on an individual, including phenotypes more proximal to the level of gene action such as levels of gene expression, microRNAs, or metabolic and proteomic profiles.