Inter-Laboratory Evaluation of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed.

METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance.

RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected.

CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.

Understanding next generation sequencing in oncology: A guide for oncologists

DNA sequencing is now faster and cheaper than ever before, due to the development of next generation sequencing (NGS) technologies. NGS is now widely used in the research setting and is becoming increasingly utilised in clinical practice. However, due to evolving clinical commitments, increased workload and lack of training opportunities, many oncologists may be unfamiliar with the terminology and technology involved. This can lead to oncologists feeling daunted by issues such as how to interpret the vast amounts of data generated by NGS and the differences between sequencing platforms. This review article explains common concepts and terminology, summarises the process of DNA sequencing (including data analysis) and discusses the main factors to consider when deciding on a sequencing method. This article aims to improve oncologists' understanding of the most commonly used sequencing platforms and the ongoing challenges faced in expanding the use of NGS into routine clinical practice.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.

Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.

Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).

Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Next-Generation Sequencing of Hereditary Hemochromatosis-Related Genes: Novel Likely Pathogenic Variants Found in the Portuguese Population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G>C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5'-UTR of HAMP gene (c.-25G>A). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Familial amyloid polyneuropathy: TTR sequencing and "in silico" analysis

Familial amyloid polyneuropathy (FAP) or paramiloidosis is an autosomal dominant neurodegenerative disease with onset on adult age that is characterized by mutated protein deposition in the form of amyloid substance. FAP is due to a point alteration in the transthyretin (TTR) gene and until now more than 100 amyloidogenic mutations have been described in TTR gene. FAP shows a wide variation in age-at-onset (AO) (19-82 years, in Portuguese cases) and the V30M mutation often runs through several generation of asymptomatic carriers, before expressing in a proband, but the protective effect disappear in a single generation, with offspring of late-onset cases having early onset. V30M mutation does not explain alone the symptoms and AO variability of the disease observed in the same family. Our aim in this study was to identify genetic factors associated with AO variability and reduced penetrance which can have important clinical implications. To accomplish this we genotyped 230 individuals, using a directautomated sequencing approach in order to identify possible genetic modifiers within the TTR locus. After genotyping, we assessed a putative association of the SNPs found with AO and an intensive in silico analysis was performed in order to understand a possible regulation of gene expression. Although we did not find any significant association between SNPs and AO, we found very interesting and unreported results in the in silico analysis since we observed some alterations in the mechanism of splicing, transcription factors binding and miRNAs binding. All of these mechanisms when altered can lead to dysregulation of gene expression, which can have an impact in AO and phenotypic variability. These putative mechanisms of regulation of gene expression within the TTR gene could be used in the future as potential therapeutical targets, and could improve genetic counselling and follow-up of mutation carriers.

Saccharomycotin transcriptomics by next-generation sequencing

The non-standard decoding of the CUG codon in Candida cylindracea raises a number of questions about the evolutionary process of this organism and other species Candida clade for which the codon is ambiguous. In order to find some answers we studied the transcriptome of C. cylindracea, comparing its behavior with that of Saccharomyces cerevisiae (standard decoder) and Candida albicans (ambiguous decoder). The transcriptome characterization was performed using RNA-seq. This approach has several advantages over microarrays and its application is booming. TopHat and Cufflinks were the software used to build the protocol that allowed for gene quantification. About 95% of the reads were mapped on the genome. 3693 genes were analyzed, of which 1338 had a non-standard start codon (TTG/CTG) and the percentage of expressed genes was 99.4%. Most genes have intermediate levels of expression, some have little or no expression and a minority is highly expressed. The distribution profile of the CUG between the three species is different, but it can be significantly associated to gene expression levels: genes with fewer CUGs are the most highly expressed. However, CUG content is not related to the conservation level: more and less conserved genes have, on average, an equal number of CUGs. The most conserved genes are the most expressed. The lipase genes corroborate the results obtained for most genes of C. cylindracea since they are very rich in CUGs and nothing conserved. The reduced amount of CUG codons that was observed in highly expressed genes may be due, possibly, to an insufficient number of tRNA genes to cope with more CUGs without compromising translational efficiency. From the enrichment analysis, it was confirmed that the most conserved genes are associated with basic functions such as translation, pathogenesis and metabolism. From this set, genes with more or less CUGs seem to have different functions. The key issues on the evolutionary phenomenon remain unclear. However, the results are consistent with previous observations and shows a variety of conclusions that in future analyzes should be taken into consideration, since it was the first time that such a study was conducted.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Domestication and the storage starch biosynthesis pathway: Signatures of selection from a whole sorghum genome sequencing strategy

Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either ‘Landraces’ or ‘Wild and Weedy’ genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.

Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy

Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either ‘Landraces’ or ‘Wild and Weedy’ genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.