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.

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline.

Clinical impact of next-generation sequencing multi-gene panel highlighting the landscape of germline alterations in ovarian cancer patients

BRCA1 and BRCA2 are the most frequently mutated genes in ovarian cancer (OC), crucial both for the identification of cancer predisposition and therapeutic choices. However, germline variants in other genes could be involved in OC susceptibility. We characterized OC patients to detect mutations in genes other than BRCA1/2 that could be associated with a high risk to develop OC, and that could permit patients to enter the most appropriate treatment and surveillance program. Next-Generation Sequencing analysis with a 94-gene panel was performed on germline DNA of 219 OC patients. We identified 34 pathogenic/likely-pathogenic variants in BRCA1/2 and 38 in other 21 genes. Patients with pathogenic/likely-pathogenic variants in non-BRCA1/2 genes developed mainly OC alone compared to the other groups that developed also breast cancer or other tumors (p=0.001). Clinical correlation analysis showed that low-risk patients were significantly associated with platinum sensitivity (p<0.001). Regarding PARP inhibitors (PARPi) response, patients with pathogenic mutations in non-BRCA1/2 genes had significantly worse PFS and OS. Moreover, a statistically significant worse PFS was found for every increase of one thousand platelets before PARPi treatment. To conclude, knowledge about molecular alterations in genes beyond BRCA1/2 in OC could allow for more personalized diagnostic, predictive, prognostic, and therapeutic strategies for OC patients.

Diversity of zooplankton assemblages at Station L4, Western English Channel, measured using next generation DNA sequencing

Background: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel Next Generation Sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify the richness and diversity of a mixed zooplankton assemblage from a productive monitoring site in the Western English Channel. Methodology/Principle Findings: Plankton WP2 replicate net hauls (200 µm) were taken at the Western Channel Observatory long-term monitoring station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,042 sequences were obtained for all samples. The sequences clustered in to 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 138 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 75 taxonomic groups. Conclusions: The percentage of OTUs assigned to major eukaryotic taxonomic groups broadly aligns between the metagenetic and morphological analysis and are dominated by Copepoda. However, the metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for estimating diversity and species richness of zooplankton communities.

'Next generation' biogeography: towards understanding the drivers of species diversification and persistence

The drivers of species diversification and persistence are of great interest to current biogeography, especially in those global biodiversity hotspots' harbouring most of Earth's animal and plant life. Classical multispecies biogeographical work has yielded fascinating insights into broad-scale patterns of diversification, and DNA-based intraspecific phylogeographical studies have started to complement this picture at much finer temporal and spatial scales. The advent of novel next-generation sequencing (NGS) technologies provides the opportunity to greatly scale up the numbers of individuals, populations and species sampled, potentially merging intraspecific and interspecific approaches to biogeographical inference. Here, we outline these prospects and issues by using the example of an undisputed hotspot, the Cape of southern Africa. We outline the current state of knowledge on the biogeography of species diversification within the Cape, review the literature for phylogeographical evidence of its likely drivers and mechanisms, and suggest possible ways forward based on NGS approaches. We demonstrate the potential of these methods and current bioinformatic issues with the help of restriction-site-associated DNA (RAD) sequencing data for three highly divergent species of the Restionaceae, an important plant radiation in the Cape. A thorough understanding of the mechanisms that facilitate species diversification and persistence in spatially structured, species-rich environments will require the adoption of novel genomic and bioinformatic tools in biogeographical studies.

Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.

Next generation e-learning platform: virtual laboratory simulations

Bioscience subjects require a significant amount of training in laboratory techniques to produce highly skilled science graduates. Many techniques which are currently used in diagnostic, research and industrial laboratories require expensive equipment for single users; examples of which include next generation sequencing, quantitative PCR, mass spectrometry and other analytical techniques. The cost of the machines, reagents and limited access frequently preclude undergraduate students from using such cutting edge techniques. In addition to cost and availability, the time taken for analytical runs on equipment such as High Performance Liquid Chromatography (HPLC) does not necessarily fit with the limitations of timetabling. Understanding the theory underlying these techniques without the accompanying practical classes can be unexciting for students. One alternative from wet laboratory provision is to use virtual simulations of such practical which enable students to see the machines and interact with them to generate data. The Faculty of Science and Technology at the University of Westminster has provided all second and third year undergraduate students with iPads so that these students all have access to a mobile device to assist with learning. We have purchased licences from Labster to access a range of virtual laboratory simulations. These virtual laboratories are fully equipped and require student responses to multiple answer questions in order to progress through the experiment. In a pilot study to look at the feasibility of the Labster virtual laboratory simulations with the iPad devices; second year Biological Science students (n=36) worked through the Labster HPLC simulation on iPads. The virtual HPLC simulation enabled students to optimise the conditions for the separation of drugs. Answers to Multiple choice questions were necessary to progress through the simulation, these focussed on the underlying principles of the HPLC technique. Following the virtual laboratory simulation students went to a real HPLC in the analytical suite in order to separate of asprin, caffeine and paracetamol. In a survey 100% of students (n=36) in this cohort agreed that the Labster virtual simulation had helped them to understand HPLC. In free text responses one student commented that "The terminology is very clear and I enjoyed using Labster very much”. One member of staff commented that “there was a very good knowledge interaction with the virtual practical”.

Microsatellite markers for Bokermannohyla species (Anura, Hylidae) from the Brazilian Cerrado and Atlantic Forest domains

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Novel and cross-amplified microsatellite loci for the critically endangered São Paulo marsh antwren Formicivora paludicola (Aves: Thamnophilidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microsatellite loci in the oceanic whitetip shark and cross-species amplification using pyrosequencing technology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ecological genetics and conservation genomics of wolf (Canis lupus)

In the present work, we apply both traditional and Next Generation Sequencing (NGS) tools to investigate some of the most important adaptive traits of wolves (Canis lupus). In the first part, we analyze the variability of three Major Histocompatibility Complex (MHC) class II genes in the Italian wolf population, also studying their possible role in mating choice and their influence on fitness traits. In the second section, as part of a larger canid genome project, we will exploit NGS data to investigate the transcript-level differences between the wolf and the dog genome that can be correlated to domestication.

Whole genome sequencing as a means to assess pathogenic mutations in medical genetics and cancer.

The past decade has seen the emergence of next-generation sequencing (NGS) technologies, which have revolutionized the field of human molecular genetics. With NGS, significant portions of the human genome can now be assessed by direct sequence analysis, highlighting normal and pathological variants of our DNA. Recent advances have also allowed the sequencing of complete genomes, by a method referred to as whole genome sequencing (WGS). In this work, we review the use of WGS in medical genetics, with specific emphasis on the benefits and the disadvantages of this technique for detecting genomic alterations leading to Mendelian human diseases and to cancer.

Strategies for the Next Generation of Conservation: Lessons from a Life in Environmental Leadership

Successful leadership in the conservation community depends upon leaders with a vision who can engage collaborators in a transaction to create something of enduring value. Ideally, results are achieved in a win-win fashion with other stakeholders. However, a good leader must employ a broader range of tools.

Characterization, development and multiplexing of microsatellite markers in three commercially exploited reef fish and their application for stock identification

Thirty-four microsatellite loci were isolated from three reef fish species; golden snapper Lutjanus johnii, blackspotted croaker Protonibea diacanthus and grass emperor Lethrinus laticaudis using a next generation sequencing approach. Both IonTorrent single reads and Illumina MiSeq paired-end reads were used, with the latter demonstrating a higher quality of reads than the IonTorrent. From the 1–1.5 million raw reads per species, we successfully obtained 10–13 polymorphic loci for each species, which satisfied stringent design criteria. We developed multiplex panels for the amplification of the golden snapper and the blackspotted croaker loci, as well as post-amplification pooling panels for the grass emperor loci. The microsatellites characterized in this work were tested across three locations of northern Australia. The microsatellites we developed can detect population differentiation across northern Australia and may be used for genetic structure studies and stock identification.