Morph-specific fitness throughout the life cycle of the grain aphid, nonhost-alternating, holocyclic Sitobion avenae (Hemiptera: Aphididae).

Aphids exhibit seasonally alternating asexual and sexual reproductive modes. Different morphs are produced throughout the life cycle. To evaluate morph-specific fitness during reproductive switching, holocyclic Sitobion avenae were induced continuously under short light conditions, and development and reproduction were compared in each morph. Seven morphs, including apterous and alate virginoparae, apterous and alate sexuparae, oviparae, males, and fundatrices, were produced during the life cycle. The greatest proportions of sexuparae, oviparae, males, and virginoparae were in the G1, G2, G3, and G4 generations, respectively. Regardless of asexual or sexual morphs, alate morphs exhibited a marked delay in age at maturity compared with that of apterous morphs. Among the alate morphs, males had the longest age at maturity, followed by sexuparae and virginoparae. Among the apterous morphs, sexuparae were older at maturity than the fundatrices, virginoparae, and oviparae. The nymphs of each morph had equal survival potentials. For the same wing morphs, apterous sexuparae and oviparae exhibited substantial delays in the pre-reproductive period and considerable reductions in fecundity, compared with those of apterous virginoparae and fundatrices, whereas alate sexuparae and alate virginoparae had similar fecundity. The seven morphs exhibited Deevey I survivorship throughout the life cycle. These results suggest that sexual production, particularly in males, has short-term development and reproduction costs. The coexistence of sexual and asexual morphs in sexuparae offspring may be regarded as an adaptive strategy for limiting the risk of low fitness in winter.

Polyphenism in Antennal Sensilla Among Different Adult Morphs of Nonhost-Alternating, Holocyclic Sitobion avenae (Hemiptera: Aphididae).

Aphids, mainly distributed in temperate zones, exhibit seasonal generation-alternating phenomena. Across the life cycle, different morphs are produced. Sitobion avenae (Fabricius 1775) is a major pest of wheat worldwide. To elucidate olfactory perception of morph-specific behavior across their life cycle, we investigated antennal sensilla among seven morphs using scanning electron microscopy. Trichoid, placoid, coeloconic, and campaniform sensilla were identified. Trichoid sensilla, big multiporous placoid sensilla (primary rhinarium), a group of sensilla (primary rhinaria), and campaniform sensilla showed similar distribution and resemblance among morphs, whereas small multiporous placoid sensilla (secondary rhinaria) exhibited obvious differences. Compared to apterous morphs, alate morphs possessed a greater abundance of secondary rhinaria, with the greatest found in males on antennal segments III-V. Alate virginoparae and alate sexuparae ranged from six to fourteen rhinaria on antennal segment III. Fundatrices, apterous virginoparae and apterous sexuparae only had one or two secondary rhinaria on antennal segment III while they disappeared in oviparae. Secondary rhinaria, lying in a cuticle cavity, are convex or concave in their central part. In males, both forms were present, with a greater proportion of convex form than that of the concave form. Fundatrices and virginoparae had the convex form while sexuparae had the concave form. Polyphenism of secondary rhinaria might suggest their association with the olfactory functions of morph-specific behavior. These results have improved our understanding of the adaptive evolution of the antennal sensilla in nonhost-alternating, holocyclic aphids.

Long-read sequencing in deciphering human genetics to a greater depth.

Through four decades' development, DNA sequencing has inched into the era of single-molecule sequencing (SMS), or the third-generation sequencing (TGS), as represented by two distinct technical approaches developed independently by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Historically, each generation of sequencing technologies was marked by innovative technological achievements and novel applications. Long reads (LRs) are considered as the most advantageous feature of SMS shared by both PacBio and ONT to distinguish SMS from next-generation sequencing (NGS, or the second-generation sequencing) and Sanger sequencing (the first-generation sequencing). Long reads overcome the limitations of NGS and drastically improves the quality of genome assembly. Besides, ONT also contributes several unique features including ultra-long reads (ULRs) with read length above 300 kb and some close to 1 million bp, direct RNA sequencing and superior portability as made possible by pocket-sized MinION sequencer. Here, we review the history of DNA sequencing technologies and associated applications, with a special focus on the advantages as well as the limitations of ULR sequencing in genome assembly.

DNMT1-interacting RNAs block gene-specific DNA methylation.

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis.

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPß and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.

GBSA: a comprehensive software for analysing whole genome bisulfite sequencing data.

High-throughput sequencing is increasingly being used in combination with bisulfite (BS) assays to study DNA methylation at nucleotide resolution. Although several programmes provide genome-wide alignment of BS-treated reads, the resulting information is not readily interpretable and often requires further bioinformatic steps for meaningful analysis. Current post-alignment BS-sequencing programmes are generally focused on the gene-specific level, a restrictive feature when analysis in the non-coding regions, such as enhancers and intergenic microRNAs, is required. Here, we present Genome Bisulfite Sequencing Analyser (GBSA-http://ctrad-csi.nus.edu.sg/gbsa), a free open-source software capable of analysing whole-genome bisulfite sequencing data with either a gene-centric or gene-independent focus. Through analysis of the largest published data sets to date, we demonstrate GBSA's features in providing sequencing quality assessment, methylation scoring, functional data management and visualization of genomic methylation at nucleotide resolution. Additionally, we show that GBSA's output can be easily integrated with other high-throughput sequencing data, such as RNA-Seq or ChIP-seq, to elucidate the role of methylated intergenic regions in gene regulation. In essence, GBSA allows an investigator to explore not only known loci but also all the genomic regions, for which methylation studies could lead to the discovery of new regulatory mechanisms.

Feasibility of low-throughput next generation sequencing for germline DNA screening.

BACKGROUND: Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). METHODS: Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. RESULTS: The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). CONCLUSIONS: Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.

The gene signature in CCAAT-enhancer-binding protein α dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors.

C/EPBα proteins, encoded by the CCAAT-enhancer-binding protein α gene, play a crucial role in granulocytic development, and defects in this transcription factor have been reported in acute myeloid leukemia. Here, we defined the C/EBPα signature characterized by a set of genes up-regulated upon C/EBPα activation. We analyzed expression of the C/EBPα signature in a cohort of 525 patients with acute myeloid leukemia and identified a subset characterized by low expression of this signature. We referred to this group of patients as the C/EBPα dysfunctional subset. Remarkably, a large percentage of samples harboring C/EBPα biallelic mutations clustered within this subset. We hypothesize that re-activation of the C/EBPα signature in the C/EBPα dysfunctional subset could have therapeutic potential. In search for small molecules able to reverse the low expression of the C/EBPα signature we applied the connectivity map. This analysis predicted positive connectivity between the C/EBPα activation signature and histone deacetylase inhibitors. We showed that these inhibitors reactivate expression of the C/EBPα signature and promote granulocytic differentiation of primary samples from the C/EBPα dysfunctional subset harboring biallelic C/EBPα mutations. Altogether, our study identifies histone deacetylase inhibitors as potential candidates for the treatment of certain leukemias characterized by down-regulation of the C/EBPα signature.

Exome sequencing: dual role as a discovery and diagnostic tool.

Recent developments in high-throughput sequence capture methods and next-generation sequencing technologies have now made exome sequencing a viable approach to elucidate the genetic basis of Mendelian disorders with hitherto unknown etiology. In addition, exome sequencing is increasingly being employed as a diagnostic tool for specific genetic diseases, particularly in the context of those disorders characterized by significant genetic and phenotypic heterogeneity, for example, Charcot-Marie-Tooth disease and congenital disorders of glycosylation. Such disorders are challenging to interrogate with conventional polymerase chain reaction-Sanger sequencing methods, because of the inherent difficulty in prioritizing candidate genes for diagnostic testing. Here, we explore the value of exome sequencing as a diagnostic tool and discuss whether exome sequencing can come to serve a dual role in diagnosis and discovery. We summarize the current status of exome sequencing, the technical challenges facing it, and its adaptation to diagnostics, and make recommendations for the use of exome sequencing as a routine diagnostic tool. Finally, we discuss pertinent ethical concerns, such as the use of exome sequencing data, originally generated in a diagnostic context, in research investigations.

Gene discovery in familial cancer syndromes by exome sequencing: prospects for the elucidation of familial colorectal cancer type X.

Recent advances in genotyping and sequencing technologies have provided powerful tools with which to explore the genetic basis of both Mendelian (monogenic) and sporadic (polygenic) diseases. Several hundred genome-wide association studies have so far been performed to explore the genetics of various polygenic or complex diseases including those cancers with a genetic predisposition. Exome sequencing has also proven very successful in elucidating the etiology of a range of hitherto poorly understood Mendelian disorders caused by high-penetrance mutations. Despite such progress, the genetic etiology of several familial cancers, such as familial colorectal cancer type X, has remained elusive. Familial colorectal cancer type X and Lynch syndrome are similar in terms of their fulfilling certain clinical criteria, but the former group is not characterized by germline mutations in DNA mismatch-repair genes. On the other hand, the genetics of sporadic colorectal cancer have been investigated by genome-wide association studies, leading to the identification of multiple new susceptibility loci. In addition, there is increasing evidence to suggest that familial and sporadic cancers exhibit similarities in terms of their genetic etiologies. In this review, we have summarized our current knowledge of familial colorectal cancer type X, discussed current approaches to probing its genetic etiology through the application of new sequencing technologies and the recruitment of the results of colorectal cancer genome-wide association studies, and explore the challenges that remain to be overcome given the uncertainty of the current genetic model (ie, monogenic vs polygenic) of familial colorectal cancer type X.

Studying the epigenome using next generation sequencing.

The advances in next generation sequencing (NGS) technologies have had a significant impact on epigenomic research. The arrival of NGS technologies has enabled a more powerful sequencing based method--that is, ChIP-Seq--to interrogate whole genome histone modifications, improving on the conventional microarray based method (ChIP-chip). Similarly, the first human DNA methylome was mapped using NGS technologies. More importantly, studies of DNA methylation and histone modification using NGS technologies have yielded new discoveries and improved our knowledge of human biology and diseases. The concept that cytosine methylation was restricted to CpG dinucleotides has only been recently challenged by new data generated from sequencing the DNA methylome. Approximately 25% of all cytosine methylation identified in stem cells was in a non-CG context. The non-CG methylation was more enriched in gene bodies and depleted in protein binding sites and enhancers. The recent developments of third generation sequencing technologies have shown promising results of directly sequencing methylated nucleotides and having the ability to differentiate between 5-methylcytosine and 5-hydroxymethylcytosine. The importance of 5-hydroxymethylcytosine remains largely unknown, but it has been found in various tissues. 5-hydroxymethylcytosine was particularly enriched at promoters and in intragenic regions (gene bodies) but was largely absent from non-gene regions in DNA from human brain frontal lobe tissue. The presence of 5-hydroxymethylcytosine in gene bodies was more positively correlated with gene expression levels. The importance of studying 5-methylcytosine and 5-hydroxymethylcytosine separately for their biological roles will become clearer when more efficient methods to distinguish them are available.

Methylation of a single intronic CpG mediates expression silencing of the PMP24 gene in prostate cancer.

BACKGROUND: We previously demonstrated that a putative anti-tumor gene, peroxisomal membrane protein 4, 24 kDa (PMP24 or PXMP4), is silenced via DNA methylation of a CpG island in its 5' flanking region (5'-CGI) in prostate cancer (PCa) cells. METHODS: To identify demethylation hypersensitive site(s) in PMP24 5'-CGI, PC-3 cells with methylated 5'-CGI were treated with a low-dose of 5-aza-2'-deoxycytidine (5-aza-dC) just sufficient to reactivate gene expression, referred as the limited demethylation approach. Gel shift assays and promoter analyzes were performed to demonstrate the role of the hypersensitive site in PMP24 gene regulation. Transfection of a methylated oligonucleotide corresponding to the hypersensitive site was conducted to determine the effect of site-specific methylation on the gene expression. Bisulfite sequencing analysis was performed to reveal the methylation status of PMP24 promoter in cultured cells and microdissected samples. In situ hybridization was applied to determine expression positivity of PMP24 mRNA. RESULTS: A 5-aza-dC hypersensitive site encompasses two CpG dinucleotides in intron 1 was identified. Methylation of the first, but not the second, CpG dinucleotide of this site disrupted DNA-protein interactions and suppressed the gene expression. Using archival specimens, we found the first CpG dinucleotide of the hypersensitive site is hypermethylated with a loss of PMP24 mRNA expression in microdissected PCa cells when compared to normal prostatic epithelial cells. CONCLUSIONS: These findings support a critical role for a single intronic CpG dinucleotide in PMP24 gene regulation through DNA methylation. The data suggest that methylation-mediated silencing of PMP24 is a molecular event associated with prostate carcinogenesis.

A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing.

BACKGROUND: We describe a novel system control (SC) implemented in an automated AmpliSeq™-based next-generation sequencing (NGS)2 run that simultaneously acts as (a) an external positive/sensitivity control, (b) a spike-in QC for DNA extraction, and (c) a nontemplate control to detect exogenous DNA contamination. METHODS: Plasmids carrying wild-type tobacco mosaic virus sequence and a sequence with three designed mutations were synthesized and mixed, such that the mutations are present at 5% variant frequency in the mixture designated as SC. SC was used as a stand-alone sample and spiked into each sample in each run. A cell line-derived reference material, in both a formalin-fixed paraffin-embedded (FFPE) sample and genomic DNA (gDNA), was sequenced in the same runs. RESULTS: By interpolation, 100 fg SC spiked in FFPE sample produced sequencing coverage equivalent to approximately 3 fg in the gDNA. In the SC-only sample, all three designed mutations were recovered around 5% as expected, while no significant reads of human genome were present. In samples with a common PCR inhibitor, coverage for both SC and target amplicons were eliminated. An inverse relationship between the coverage of SC and DNA input was observed. In clinical samples, the ratio of SC to the median coverage of sample can be used to indicate insufficient DNA input. CONCLUSIONS: The SC is an elegant and comprehensive QC concept for NGS-based diagnostic tests.

PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model.

Transcriptional silencing of antitumor genes via CpG island methylation could be a mechanism mediating prostate cancer (PCa) progression from an androgen-sensitive (AS) to an androgen-insensitive (AI) state. We have used the methylation-sensitive restriction fingerprinting (MSRF) technique to identify novel CpG-rich sequences that are differentially methylated between the genome of the AS PCa cell line LNCaP and that of an AI subline LNCaP(CS) generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. One such sequence identified was located on a 5' CpG island that was found to span part of the promoter, exon 1, and part of intron 1 of the peroxisomal membrane protein 24 kDa (PMP24) gene. Using semiquantitative RT-PCR and bisulfite genomic sequencing, we established an inverse relationship between mRNA expression and methylation of the 5' CpG island of PMP24. PMP24 mRNA was absent in LNCaP(CS) and the androgen receptor-negative PC-3 cell line; both exhibited dense methylation in the said CpG island. In contrast, PMP24 mRNA was expressed in LNCaP and normal prostatic epithelial cells (NPrECs) whose PMP24 5' CpG island remained unmethylated. Treatment of LNCaP(CS) and PC-3 with the demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) reactivated PMP24 mRNA expression. Transient transfection of PMP24 into LNCaP(CS) and PC-3 cells induced a significant reduction in cell growth and soft-agar colony formation potential, suggesting that PMP24 gene product has antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence.

The novel estrogen 17alpha-20Z-21-[(4-amino)phenyl]-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diol induces apoptosis in prostate cancer cell lines at nanomolar concentrations in vitro.

Prostate cancer remains the number one cause of noncutaneous cancer, with 220,900 new cases predicted for the year 2003 alone. Of the more promising classes of compounds studied thus far for the treatment of prostate cancer, estrogens of various types have consistently exhibited antitumor activities both in vitro and in vivo. For this reason, we have synthesized and screened a library of unique 17alpha/11beta modified 17beta-estradiol (E(2)) analogues designed for estrogen receptor beta (ER-beta) specificity and a potential for cytotoxic activity directed toward prostate cancer cells. From this library, the novel compound 17alpha-20Z-21-[(4-amino)phenyl]-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diol (APVE(2)) was identified as the primary lead, found to induce a high level (>90%) of cell death through an apoptotic mechanism, with an EC(50) of 1.4, 2.7, and 16 nM in the LNCaP, PC3, and DU145 cell lines, respectively. APVE(2) was found to bind to ER-beta, albeit weakly, with an EC(50) of 250 nM and a binding activity of 6.2% relative to E(2), nearly two orders of magnitude less than the concentration required to induce apoptosis. APVE(2) bound preferentially to ER-beta by 7-fold over ER-alpha, and did not induce growth in the MCF-7 cell line, thus indicating that it is not a classical ER agonist. Furthermore, the cytotoxic actions of APVE(2) were not reversed by co-treatment with a 50-fold excess E(2). In summary, a novel 17 modified estrogen APVE(2) was identified as a lead compound, capable of inducing apoptosis in three prostate cancer cell lines at low nanomolar concentrations, through a mechanism inconsistent with an ER-mediated mechanism.

Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

Chemotherapy-induced monoamine oxidase expression in prostate carcinoma functions as a cytoprotective resistance enzyme and associates with clinical outcomes.

To identify molecular alterations in prostate cancers associating with relapse following neoadjuvant chemotherapy and radical prostatectomy patients with high-risk localized prostate cancer were enrolled into a phase I-II clinical trial of neoadjuvant chemotherapy with docetaxel and mitoxantrone followed by prostatectomy. Pre-treatment prostate tissue was acquired by needle biopsy and post-treatment tissue was acquired by prostatectomy. Prostate cancer gene expression measurements were determined in 31 patients who completed 4 cycles of neoadjuvant chemotherapy. We identified 141 genes with significant transcript level alterations following chemotherapy that associated with subsequent biochemical relapse. This group included the transcript encoding monoamine oxidase A (MAOA). In vitro, cytotoxic chemotherapy induced the expression of MAOA and elevated MAOA levels enhanced cell survival following docetaxel exposure. MAOA activity increased the levels of reactive oxygen species and increased the expression and nuclear translocation of HIF1α. The suppression of MAOA activity using the irreversible inhibitor clorgyline augmented the apoptotic responses induced by docetaxel. In summary, we determined that the expression of MAOA is induced by exposure to cytotoxic chemotherapy, increases HIF1α, and contributes to docetaxel resistance. As MAOA inhibitors have been approved for human use, regimens combining MAOA inhibitors with docetaxel may improve clinical outcomes.

Dynamic analysis of gene expression and genome-wide transcription factor binding during lineage specification of multipotent progenitors.

We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.

Technological advances in DNA sequence enrichment and sequencing for germline genetic diagnosis.

The potential applications of next-generation sequencing technologies in diagnostic laboratories have become increasingly evident despite the various technical challenges that still need to be overcome to potentiate its widespread adoption in a clinical setting. Whole-genome sequencing is now both technically feasible and 'cost effective' using next-generation sequencing techniques. However, this approach is still considered to be 'expensive' for a diagnostic test. Although the goal of the US$1000 genome is fast approaching, neither the analytical hurdles nor the ethical issues involved are trivial. In addition, the cost of data analysis and storage has been much higher than initially expected. As a result, it is widely perceived that targeted sequencing and whole-exome sequencing are more likely to be adopted as diagnostic tools in the foreseeable future. However, the information-generating power of whole-exome sequencing has also sparked considerable debate in relation to its deployment in genetic diagnostics, particularly with reference to the revelation of incidental findings. In this review, we focus on the targeted sequencing approach and its potential as a genetic diagnostic tool.

Exome versus transcriptome sequencing in identifying coding region variants.

The advent of next-generation sequencing technologies has revolutionized the study of genetic variation in the human genome. Whole-genome sequencing currently represents the most comprehensive strategy for variant detection genome-wide but is costly for large sample sizes, and variants detected in noncoding regions remain largely uninterpretable. By contrast, whole-exome sequencing has been widely applied in the identification of germline mutations underlying Mendelian disorders, somatic mutations in various cancers and de novo mutations in neurodevelopmental disorders. Since whole-exome sequencing focuses upon the entire set of exons in the genome (the exome), it requires additional exome-enrichment steps compared with whole-genome sequencing. Although the availability of multiple commercial exome-enrichment kits has made whole-exome sequencing technically feasible, it has also added to the overall cost. This has led to the emergence of transcriptome (or RNA) sequencing as a potential alternative approach to variant detection within protein coding regions, since the transcriptome of a given tissue represents a quasi-complete set of transcribed genes (mRNAs) and other noncoding RNAs. A further advantage of this approach is that it bypasses the need for exome enrichment. Here we discuss the relative merits and limitations of these approaches as they are applied in the context of variant detection within gene coding regions.