Sources of PCR-induced distortions in high-throughput sequencing data sets

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules

A Security Monitoring Framework For Virtualization Based HEP Infrastructures

High Energy Physics (HEP) distributed computing infrastructures require automatic tools to monitor, analyze and react to potential security incidents. These tools should collect and inspect data such as resource consumption, logs and sequence of system calls for detecting anomalies that indicate the presence of a malicious agent. They should also be able to perform automated reactions to attacks without administrator intervention. We describe a novel framework that accomplishes these requirements, with a proof of concept implementation for the ALICE experiment at CERN. We show how we achieve a fully virtualized environment that improves the security by isolating services and Jobs without a significant performance impact. We also describe a collected dataset for Machine Learning based Intrusion Prevention and Detection Systems on Grid computing. This dataset is composed of resource consumption measurements (such as CPU, RAM and network traffic), logfiles from operating system services, and system call data collected from production Jobs running in an ALICE Grid test site and a big set of malware. This malware was collected from security research sites. Based on this dataset, we will proceed to develop Machine Learning algorithms able to detect malicious Jobs.Comment: Proceedings of the 22nd International Conference on Computing in High Energy and Nuclear Physics, CHEP 2016, 10-14 October 2016, San Francisco. Submitted to Journal of Physics: Conference Series (JPCS

Genexpressionsprofile humaner intestinaler mikrovaskulärer Endothelzellen (HIMEC)

In dieser Arbeit wurde das Genexpressionsprofil von HIMEC sowie seine Modulation durch das proinflammatorische Zytokin TNF-alpha charakterisiert. Die Genexpression TNF-alpha stimulierter HIMEC wurde mittels Gene Arrays analysiert und mit der der mikrovaskulären Referenz-Endothelzellinie HMEC-1 verglichen. Von den nach Stimulation hochregulierten Genen sind nur < 50% in beiden Zellpopulationen anzutreffen. Es zeigen sich in HIMEC mehrere bisher unbekannte Genexpressionsmuster sowie einige bislang für Endothelzellen bzw. intestinales Gewebe unbeschriebene hochregulierte Gene. Die in HIMEC durch den proinflammatorischen Mediator TNF-alpha differenziell regulierten neuen Kandidatengene spielen möglicherweise im Rahmen der mukosalen Entzündung eine bislang unbekannte Rolle

Rosetta Brains: A Strategy for Molecularly-Annotated Connectomics

We propose a neural connectomics strategy called Fluorescent In-Situ Sequencing of Barcoded Individual Neuronal Connections (FISSEQ-BOINC), leveraging fluorescent in situ nucleic acid sequencing in fixed tissue (FISSEQ). FISSEQ-BOINC exhibits different properties from BOINC, which relies on bulk nucleic acid sequencing. FISSEQ-BOINC could become a scalable approach for mapping whole-mammalian-brain connectomes with rich molecular annotations

Using high-throughput barcode sequencing to efficiently map connectomes

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost

BSP implementation of European S3 - level evidence-based treatment guidelines for stage I-III periodontitis in UK clinical practice

OBJECTIVES: To adapt the supranational European Federation for Periodontology (EFP) S3-Level Clinical Practice Guideline for treatment of periodontitis (stage I-III) to a UK healthcare environment, taking into account the views of a broad range of stakeholders, and patients. SOURCES: This UK version is based on the supranational EFP guideline (Sanz et al., 2020) published in the Journal of Clinical Periodontology. The source guideline was developed using the S3-level methodology, which combined the assessment of formal evidence from 15 systematic reviews with a moderated consensus process of a representative group of stakeholders, and accounts for health equality, environmental factors and clinical effectiveness. It encompasses 62 clinical recommendations for the treatment of stage I-III periodontitis, based on a step-wise process mapped to the 2017 classification system. METHODOLOGY: The UK version was developed from the source guideline using a formal process called the GRADE ADOLOPMENT framework. This framework allows for the adoption (unmodified acceptance), adaptation (acceptance with modifications) and the de novo development of clinical recommendations. Using this framework and following the S3-process, the underlying systematic reviews were updated and a representative guideline group of 75 delegates from 17 stakeholder organisations was assembled into three working groups. Following the formal S3-process, all clinical recommendations were formally assessed for their applicability to the UK and adoloped accordingly. RESULTS AND CONCLUSION: Using the ADOLOPMENT protocol, a UK version of the EFP S3-level clinical practice guideline was developed. This guideline delivers evidence- and consensus-based clinical recommendations of direct relevance to the dental community in the UK. CLINICAL SIGNIFICANCE: The aim of S3-level guidelines is to combine the evaluation of formal evidence, grading and synthesis with the clinical expertise of a broad range of stakeholders to form clinical recommendations. Herein, the first major international S3-level guideline in dentistry, the EFP guideline, was implemented for direct clinical applicability in the UK healthcare system

Prevention and treatment of peri-implant diseases-The EFP S3 level clinical practice guideline.

BACKGROUND: The recently published Clinical Practice Guidelines (CPGs) for the treatment of stages I-IV periodontitis provided evidence-based recommendations for treating periodontitis patients, defined according to the 2018 classification. Peri-implant diseases were also re-defined in the 2018 classification. It is well established that both peri-implant mucositis and peri-implantitis are highly prevalent. In addition, peri-implantitis is particularly challenging to manage and is accompanied by significant morbidity. AIM: To develop an S3 level CPG for the prevention and treatment of peri-implant diseases, focusing on the implementation of interdisciplinary approaches required to prevent the development of peri-implant diseases or their recurrence, and to treat/rehabilitate patients with dental implants following the development of peri-implant diseases. MATERIALS AND METHODS: This S3 level CPG was developed by the European Federation of Periodontology, following methodological guidance from the Association of Scientific Medical Societies in Germany and the Grading of Recommendations Assessment, Development and Evaluation process. A rigorous and transparent process included synthesis of relevant research in 13 specifically commissioned systematic reviews, evaluation of the quality and strength of evidence, formulation of specific recommendations, and a structured consensus process involving leading experts and a broad base of stakeholders. RESULTS: The S3 level CPG for the prevention and treatment of peri-implant diseases culminated in the recommendation for implementation of various different interventions before, during and after implant placement/loading. Prevention of peri-implant diseases should commence when dental implants are planned, surgically placed and prosthetically loaded. Once the implants are loaded and in function, a supportive peri-implant care programme should be structured, including periodical assessment of peri-implant tissue health. If peri-implant mucositis or peri-implantitis are detected, appropriate treatments for their management must be rendered. CONCLUSION: The present S3 level CPG informs clinical practice, health systems, policymakers and, indirectly, the public on the available and most effective modalities to maintain healthy peri-implant tissues, and to manage peri-implant diseases, according to the available evidence at the time of publication

Multiplicity dependence of jet-like two-particle correlations in p-Pb collisions at sNN\sqrt{s_{NN}} = 5.02 TeV

Two-particle angular correlations between unidentified charged trigger and associated particles are measured by the ALICE detector in p-Pb collisions at a nucleon-nucleon centre-of-mass energy of 5.02 TeV. The transverse-momentum range 0.7 $< p_{\rm{T}, assoc} < p_{\rm{T}, trig} <$ 5.0 GeV/$c$ is examined, to include correlations induced by jets originating from low momen\-tum-transfer scatterings (minijets). The correlations expressed as associated yield per trigger particle are obtained in the pseudorapidity range $|\eta|<0.9$. The near-side long-range pseudorapidity correlations observed in high-multiplicity p-Pb collisions are subtracted from both near-side short-range and away-side correlations in order to remove the non-jet-like components. The yields in the jet-like peaks are found to be invariant with event multiplicity with the exception of events with low multiplicity. This invariance is consistent with the particles being produced via the incoherent fragmentation of multiple parton--parton scatterings, while the yield related to the previously observed ridge structures is not jet-related. The number of uncorrelated sources of particle production is found to increase linearly with multiplicity, suggesting no saturation of the number of multi-parton interactions even in the highest multiplicity p-Pb collisions. Further, the number scales in the intermediate multiplicity region with the number of binary nucleon-nucleon collisions estimated with a Glauber Monte-Carlo simulation.Comment: 23 pages, 6 captioned figures, 1 table, authors from page 17, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/161

Multi-particle azimuthal correlations in p-Pb and Pb-Pb collisions at the CERN Large Hadron Collider

Measurements of multi-particle azimuthal correlations (cumulants) for charged particles in p-Pb and Pb-Pb collisions are presented. They help address the question of whether there is evidence for global, flow-like, azimuthal correlations in the p-Pb system. Comparisons are made to measurements from the larger Pb-Pb system, where such evidence is established. In particular, the second harmonic two-particle cumulants are found to decrease with multiplicity, characteristic of a dominance of few-particle correlations in p-Pb collisions. However, when a $|\Delta \eta|$ gap is placed to suppress such correlations, the two-particle cumulants begin to rise at high-multiplicity, indicating the presence of global azimuthal correlations. The Pb-Pb values are higher than the p-Pb values at similar multiplicities. In both systems, the second harmonic four-particle cumulants exhibit a transition from positive to negative values when the multiplicity increases. The negative values allow for a measurement of $v_{2}\{4\}$ to be made, which is found to be higher in Pb-Pb collisions at similar multiplicities. The second harmonic six-particle cumulants are also found to be higher in Pb-Pb collisions. In Pb-Pb collisions, we generally find $v_{2}\{4\} \simeq v_{2}\{6\}\neq 0$ which is indicative of a Bessel-Gaussian function for the $v_{2}$ distribution. For very high-multiplicity Pb-Pb collisions, we observe that the four- and six-particle cumulants become consistent with 0. Finally, third harmonic two-particle cumulants in p-Pb and Pb-Pb are measured. These are found to be similar for overlapping multiplicities, when a $|\Delta\eta| > 1.4$ gap is placed.Comment: 25 pages, 11 captioned figures, 3 tables, authors from page 20, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/87