Improving Short DNA Sequence Alignment with Parallel Computing

Variations in different types of genomes have been found to be responsible for a large degree of physical diversity such as appearance and susceptibility to disease. Identification of genomic variations is difficult and can be facilitated through computational analysis of DNA sequences. Newly available technologies are able to sequence billions of DNA base pairs relatively quickly. These sequences can be used to identify variations within their specific genome but must be mapped to a reference sequence first. In order to align these sequences to a reference sequence, we require mapping algorithms that make use of approximate string matching and string indexing methods. To date, few mapping algorithms have been tailored to handle the massive amounts of output generated by newly available sequencing technologies. In otrder to handle this large amount of data, we modified the popular mapping software BWA to run in parallel using OpenMPI. Parallel BWA matches the efficiency of multithreaded BWA functions while providing efficient parallelism for BWA functions that do not currently support multithreading. Parallel BWA shows significant wall time speedup in comparison to multithreaded BWA on high-performance computing clusters, and will thus facilitate the analysis of genome sequencing data.

Automated parallel DNA sequencing on multiple channel microchips

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Highly parallel genetic approaches in Mendelian and complex diseases

The recent advance in high-throughput sequencing and genotyping protocols allows rapid investigation of Mendelian and complex diseases on a scale not previously been possible. In my thesis research I took advantage of these modern techniques to study retinitis pigmentosa (RP), a rare inherited disease characterized by progressive loss of photoreceptors and leading to blindness; and hypertension, a common condition affecting 30% of the adult population. Firstly, I compared the performance of different next generation sequencing (NGS) platforms in the sequencing of the RP-linked gene PRPF31. The gene contained a mutation in an intronic repetitive element, which presented difficulties for both classic sequencing methods and NGS. We showed that all NGS platforms are powerful tools to identify rare and common DNA variants, also in case of more complex sequences. Moreover, we evaluated the features of different NGS platforms that are important in re-sequencing projects. The main focus of my thesis was then to investigate the involvement of pre-mRNA splicing factors in autosomal dominant RP (adRP). I screened 5 candidate genes in a large cohort of patients by using long-range PCR as enrichment step, followed by NGS. We tested two different approaches: in one, all target PCRs from all patients were pooled and sequenced as a single DNA library; in the other, PCRs from each patient were separated within the pool by DNA barcodes. The first solution was more cost-effective, while the second one allowed obtaining faster and more accurate results, but overall they both proved to be effective strategies for gene screenings in many samples. We could in fact identify novel missense mutations in the SNRNP200 gene, encoding an essential RNA helicase for splicing catalysis. Interestingly, one of these mutations showed incomplete penetrance in one family with adRP. Thus, we started to study the possible molecular causes underlying phenotypic differences between asymptomatic and affected members of this family. For the study of hypertension, I joined a European consortium to perform genome-wide association studies (GWAS). Thanks to the use of very informative genotyping arrays and of phenotipically well-characterized cohorts, we could identify a novel susceptibility locus for hypertension in the promoter region of the endothelial nitric oxide synthase gene (NOS3). Moreover, we have proven the direct causality of the associated SNP using three different methods: 1) targeted resequencing, 2) luciferase assay, and 3) population study. - Le récent progrès dans le Séquençage à haut Débit et les protocoles de génotypage a permis une plus vaste et rapide étude des maladies mendéliennes et multifactorielles à une échelle encore jamais atteinte. Durant ma thèse de recherche, j'ai utilisé ces nouvelles techniques de séquençage afin d'étudier la retinite pigmentale (RP), une maladie héréditaire rare caractérisée par une perte progressive des photorécepteurs de l'oeil qui entraine la cécité; et l'hypertension, une maladie commune touchant 30% de la population adulte. Tout d'abord, j'ai effectué une comparaison des performances de différentes plateformes de séquençage NGS (Next Generation Sequencing) lors du séquençage de PRPF31, un gène lié à RP. Ce gène contenait une mutation dans un élément répétable intronique, qui présentait des difficultés de séquençage avec la méthode classique et les NGS. Nous avons montré que les plateformes de NGS analysées sont des outils très puissants pour identifier des variations de l'ADN rares ou communes et aussi dans le cas de séquences complexes. De plus, nous avons exploré les caractéristiques des différentes plateformes NGS qui sont importantes dans les projets de re-séquençage. L'objectif principal de ma thèse a été ensuite d'examiner l'effet des facteurs d'épissage de pre-ARNm dans une forme autosomale dominante de RP (adRP). Un screening de 5 gènes candidats issus d'une large cohorte de patients a été effectué en utilisant la long-range PCR comme étape d'enrichissement, suivie par séquençage avec NGS. Nous avons testé deux approches différentes : dans la première, toutes les cibles PCRs de tous les patients ont été regroupées et séquencées comme une bibliothèque d'ADN unique; dans la seconde, les PCRs de chaque patient ont été séparées par code barres d'ADN. La première solution a été la plus économique, tandis que la seconde a permis d'obtenir des résultats plus rapides et précis. Dans l'ensemble, ces deux stratégies se sont démontrées efficaces pour le screening de gènes issus de divers échantillons. Nous avons pu identifier des nouvelles mutations faux-sens dans le gène SNRNP200, une hélicase ayant une fonction essentielle dans l'épissage. Il est intéressant de noter qu'une des ces mutations montre une pénétrance incomplète dans une famille atteinte d'adRP. Ainsi, nous avons commencé une étude sur les causes moléculaires entrainant des différences phénotypiques entre membres affectés et asymptomatiques de cette famille. Lors de l'étude de l'hypertension, j'ai rejoint un consortium européen pour réaliser une étude d'association Pangénomique ou genome-wide association study Grâce à l'utilisation de tableaux de génotypage très informatifs et de cohortes extrêmement bien caractérisées au niveau phénotypique, un nouveau locus lié à l'hypertension a été identifié dans la région promotrice du gène endothélial nitric oxide sinthase (NOS3). Par ailleurs, nous avons prouvé la cause directe du SNP associé au moyen de trois méthodes différentes: i) en reséquençant la cible avec NGS, ii) avec des essais à la luciférase et iii) une étude de population.

PMCRI: a parallel modular classification rule induction framework

In a world where massive amounts of data are recorded on a large scale we need data mining technologies to gain knowledge from the data in a reasonable time. The Top Down Induction of Decision Trees (TDIDT) algorithm is a very widely used technology to predict the classification of newly recorded data. However alternative technologies have been derived that often produce better rules but do not scale well on large datasets. Such an alternative to TDIDT is the PrismTCS algorithm. PrismTCS performs particularly well on noisy data but does not scale well on large datasets. In this paper we introduce Prism and investigate its scaling behaviour. We describe how we improved the scalability of the serial version of Prism and investigate its limitations. We then describe our work to overcome these limitations by developing a framework to parallelise algorithms of the Prism family and similar algorithms. We also present the scale up results of a first prototype implementation.

Scaling up data mining techniques to large datasets using parallel and distributed processing

Advances in hardware and software technology enable us to collect, store and distribute large quantities of data on a very large scale. Automatically discovering and extracting hidden knowledge in the form of patterns from these large data volumes is known as data mining. Data mining technology is not only a part of business intelligence, but is also used in many other application areas such as research, marketing and financial analytics. For example medical scientists can use patterns extracted from historic patient data in order to determine if a new patient is likely to respond positively to a particular treatment or not; marketing analysts can use extracted patterns from customer data for future advertisement campaigns; finance experts have an interest in patterns that forecast the development of certain stock market shares for investment recommendations. However, extracting knowledge in the form of patterns from massive data volumes imposes a number of computational challenges in terms of processing time, memory, bandwidth and power consumption. These challenges have led to the development of parallel and distributed data analysis approaches and the utilisation of Grid and Cloud computing. This chapter gives an overview of parallel and distributed computing approaches and how they can be used to scale up data mining to large datasets.

Dynamic group communication for large-scale parallel data mining

Exascale systems are the next frontier in high-performance computing and are expected to deliver a performance of the order of 10^18 operations per second using massive multicore processors. Very large- and extreme-scale parallel systems pose critical algorithmic challenges, especially related to concurrency, locality and the need to avoid global communication patterns. This work investigates a novel protocol for dynamic group communication that can be used to remove the global communication requirement and to reduce the communication cost in parallel formulations of iterative data mining algorithms. The protocol is used to provide a communication-efficient parallel formulation of the k-means algorithm for cluster analysis. The approach is based on a collective communication operation for dynamic groups of processes and exploits non-uniform data distributions. Non-uniform data distributions can be either found in real-world distributed applications or induced by means of multidimensional binary search trees. The analysis of the proposed dynamic group communication protocol has shown that it does not introduce significant communication overhead. The parallel clustering algorithm has also been extended to accommodate an approximation error, which allows a further reduction of the communication costs. The effectiveness of the exact and approximate methods has been tested in a parallel computing system with 64 processors and in simulations with 1024 processing elements.

Massive Open Online Courses on health and medicine: review

Background: Massive Open Online Courses (MOOCs) have become immensely popular in a short span of time. However, there is very little research exploring MOOCs in the discipline of Health and Medicine. This paper is aimed to fill this void by providing a review of Health and Medicine related MOOCs. Objective: Provide a review of Health and Medicine related MOOCs offered by various MOOC platforms within the year 2013. Analyze and compare the various offerings, their target audience, typical length of a course and credentials offered. Discuss opportunities and challenges presented by MOOCs in the discipline of Health and Medicine. Methods: Health and Medicine related MOOCs were gathered using several methods to ensure the richness and completeness of data. Identified MOOC platform websites were used to gather the lists of offerings. In parallel, these MOOC platforms were contacted to access official data on their offerings. Two MOOC aggregator sites (Class Central and MOOC List) were also consulted to gather data on MOOC offerings. Eligibility criteria were defined to concentrate on the courses that were offered in 2013 and primarily on the subject ‘Health and Medicine’. All language translations in this paper were achieved using Google Translate. Results: The search identified 225 courses out of which 98 were eligible for the review (n = 98). 58% (57) of the MOOCs considered were offered on the Coursera platform and 94% (92) of all the MOOCs were offered in English. 90 MOOCs were offered by universities and the John Hopkins University offered the largest number of MOOCs (12). Only three MOOCs were offered by developing countries (China, West Indies, and Saudi Arabia). The duration of MOOCs varied from three weeks to 20 weeks with an average length of 6.7 weeks. On average MOOCs expected a participant to work on the material for 4.2 hours a week. Verified Certificates were offered by 14 MOOCs while three others offered other professional recognition. Conclusions: The review presents evidence to suggest that MOOCs can be used as a way to provide continuous medical education. It also shows the potential of MOOCs as a means of increasing health literacy among the public.

Elderly learners and Massive Open Online Courses: a review

Background: Massive open online courses (MOOCs) have become commonplace in the e-learning landscape. Thousands of elderly learners are participating in courses offered by various institutions on a multitude of platforms in many different languages. However, there is very little research into understanding elderly learners in MOOCs. Objective: We aim to show that a considerable proportion of elderly learners are participating in MOOCs and that there is a lack of research in this area. We hope this assertion of the wide gap in research on elderly learners in MOOCs will pave the way for more research in this area. Methods: Pre-course survey data for 10 University of Reading courses on the FutureLearn platform were analyzed to show the level of participation of elderly learners in MOOCs. Two MOOC aggregator sites (Class Central and MOOC List) were consulted to gather data on MOOC offerings that include topics relating to aging. In parallel, a selected set of MOOC platform catalogues, along with a recently published review on health and medicine-related MOOCs, were searched to find courses relating to aging. A systematic literature search was then employed to identify research articles on elderly learners in MOOCs. Results: The 10 courses reviewed had a considerable proportion of elderly learners participating in them. For the over-66 age group, this varied from 0.5% (on the course “Managing people”) to 16.3% (on the course “Our changing climate”), while for the over-56 age group it ranged from 3.0% (on “A beginners guide to writing in English”) to 39.5% (on “Heart health”). Only six MOOCs were found to include topics related to aging: three were on the Coursera platform, two on the FutureLearn platform, and one on the Open2Study platform. Just three scholarly articles relating to MOOCs and elderly learners were retrieved from the literature search. Conclusions: This review presents evidence to suggest that elderly learners are already participating in MOOCs. Despite this, there has been very little research into their engagement with MOOCs. Similarly, there has been little research into exploiting the scope of MOOCs for delivering topics that would be of interest to elderly learners. We believe there is potential to use MOOCs as a way of tackling the issue of loneliness among older adults by engaging them as either resource personnel or learners.

Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.

Influence of quantized massive matter fields on the Casimir effect

Charged massive matter fields of spin-0 and spin- 1/2 are quantized in the presence of an external uniform magnetic field in a spatial region bounded by two parallel plates. The most general set of boundary conditions at the plates, that is required by mathematical consistency and the self-adjointness of the Hamiltonian operator, is employed. The vacuum fluctuations of the matter field in the case of the magnetic field orthogonal to the plates are analyzed, and it is shown that the pressure from the vacuum onto the plates is positive and independent of the boundary condition, as well as of the distance between the plates. Possibilities of the detection of this new-type Casimir effect are discussed. Read More: http://www.worldscientific.com/doi/10.1142/S0217732315500996

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.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

Grant-free protocols for massive multiple access

In next generation Internet-of-Things, the overhead introduced by grant-based multiple access protocols may engulf the access network as a consequence of the proliferation of connected devices. Grant-free access protocols are therefore gaining an increasing interest to support massive multiple access. In addition to scalability requirements, new demands have emerged for massive multiple access, including latency and reliability. The challenges envisaged for future wireless communication networks, particularly in the context of massive access, include: i) a very large population size of low power devices transmitting short packets; ii) an ever-increasing scalability requirement; iii) a mild fixed maximum latency requirement; iv) a non-trivial requirement on reliability. To this aim, we suggest the joint utilization of grant-free access protocols, massive MIMO at the base station side, framed schemes to let the contention start and end within a frame, and succesive interference cancellation techniques at the base station side. In essence, this approach is encapsulated in the concept of coded random access with massive MIMO processing. These schemes can be explored from various angles, spanning the protocol stack from the physical (PHY) to the medium access control (MAC) layer. In this thesis, we delve into both of these layers, examining topics ranging from symbol-level signal processing to succesive interference cancellation-based scheduling strategies. In parallel with proposing new schemes, our work includes a theoretical analysis aimed at providing valuable system design guidelines. As a main theoretical outcome, we propose a novel joint PHY and MAC layer design based on density evolution on sparse graphs.