Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in ...2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy.
Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.
All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The assembly of DNA sequence data is undergoing a renaissance thanks to emerging technologies capable of producing reads tens of kilobases long. Assembling complete bacterial and small eukaryotic ...genomes is now possible, but the final step of circularizing sequences remains unsolved. Here we present Circlator, the first tool to automate assembly circularization and produce accurate linear representations of circular sequences. Using Pacific Biosciences and Oxford Nanopore data, Circlator correctly circularized 26 of 27 circularizable sequences, comprising 11 chromosomes and 12 plasmids from bacteria, the apicoplast and mitochondrion of Plasmodium falciparum and a human mitochondrion. Circlator is available at http://sanger-pathogens.github.io/circlator/ .
Methods to reliably assess the accuracy of genome sequence data are lacking. Currently completeness is only described qualitatively and mis-assemblies are overlooked. Here we present REAPR, a tool ...that precisely identifies errors in genome assemblies without the need for a reference sequence. We have validated REAPR on complete genomes or de novo assemblies from bacteria, malaria and Caenorhabditis elegans, and demonstrate that 86% and 82% of the human and mouse reference genomes are error-free, respectively. When applied to an ongoing genome project, REAPR provides corrected assembly statistics allowing the quantitative comparison of multiple assemblies. REAPR is available at http://www.sanger.ac.uk/resources/software/reapr/ .
Due to the availability of new sequencing technologies, we are now increasingly interested in sequencing closely related strains of existing finished genomes. Recently a number of de novo and ...mapping-based assemblers have been developed to produce high quality draft genomes from new sequencing technology reads. New tools are necessary to take contigs from a draft assembly through to a fully contiguated genome sequence. ABACAS is intended as a tool to rapidly contiguate (align, order, orientate), visualize and design primers to close gaps on shotgun assembled contigs based on a reference sequence. The input to ABACAS is a set of contigs which will be aligned to the reference genome, ordered and orientated, visualized in the ACT comparative browser, and optimal primer sequences are automatically generated. Availability and Implementation: ABACAS is implemented in Perl and is freely available for download from http://abacas.sourceforge.net Contact: sa4@sanger.ac.uk
Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint ...homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTK
TREM2
and MerTK
LYVE1
) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTK
STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTK
STM subpopulations could therefore be a potential treatment strategy for RA.
ACC/AHA Task Force Members Jeffrey L. Anderson, MD, FACC, FAHA, Chair, Jonathan L. Halperin, MD, FACC, FAHA, Chair-Elect, Nancy M. Albert, PhD, CCNS, CCRN, FAHA, Biykem Bozkurt, MD, PhD, FACC, FAHA, ...Ralph G. Brindis, MD, MPH, MACC, Mark A. Creager, MD, FACC, FAHA§§Task Force member during the writing effort., Lesley H. Curtis, PhD, FAHA, David DeMets, PhD, Robert A. Guyton, MD, FACC§§, Judith S. Hochman, MD, FACC, FAHA, Richard J. Kovacs, MD, FACC, FAHA, E. Magnus Ohman, MD, FACC, Susan J. Pressler, PhD, RN, FAHA, Frank W. Sellke, MD, FACC, FAHA, Win-Kuang Shen, MD, FACC, FAHA, William G. Stevenson, MD, FACC, FAHA§§, Clyde W. Yancy, MD, FACC, FAHA§§ Table of Contents Preamble... An organized and directed approach to a thorough review of evidence has resulted in the production of clinical practice guidelines that assist clinicians in selecting the best management strategy for an individual patient. ...clinical practice guidelines can provide a foundation for other applications, such as performance measures, appropriate use criteria, and both quality improvement and clinical decision support tools. ...in view of the increasing number of comparative effectiveness studies, comparator verbs and suggested phrases for writing recommendations for the comparative effectiveness of one treatment or strategy versus another are included for COR I and IIa, LOE A or B only.\n Charney Division of Cardiology; Associate Director, Health Care Center None None None None None None Craig R. Smith Content Reviewer Columbia University College of Physicians and Surgeons--Professor of Surgery; Chair, Department of Surgery; New York-Presbyterian Hospital/Columbia University Medical Center--Surgeon-in-Chief None None None Edwards Lifesciences--PI None None Ruth H. Strasser Content Reviewer--AIG Heart Centre,University Hospital, University of Technology, Dresden--Professor, Director, and Chair, Internal Medicine and Cardiology Clinic; Medical Director, Heart Centre None None None None Abbottdagger AstraZenecadagger Bayerdagger Biosensorsdagger Pfizerdagger None Rakesh Suri Content Reviewer Mayo Clinic--Associate Professor of Surgery None None None None Edwards Lifesciencesdagger Sorindagger St. Jude Medicaldagger None Vinod Thourani Content Reviewer--Surgeon Council Emory University Edward Lifesciences Sorin St. Jude Medical None Apica Cardiovasculardagger Maquet None None Alec Vahanian Content Reviewer Hospital Bichat--Department de Cardiologie Abbott Vascular Edwards Lifesciences Medtronic St. Jude Medical Valtech None None None None None Andrew Wang Content Reviewer Duke University Medical Center--Professor of Medicine None None None Abbott Vascularlow * Edwards Lifescienceslow * None Defendant, Sudden death, 2012 * This table represents the relationships of reviewers with industry and other entities that were disclosed at the time of peer review and determined to be relevant to this document. Names are listed in alphabetical order within each category of review.According to the ACC/AHA, a person has a relevant relationship IF: a) The relationship or interest relates to the same or similar subject matter, intellectual property or asset, topic, or issue addressed in the document; or b) The company/entity (with whom the relationship exists) makes a drug, drug class, or device addressed in the document, or makes a competing drug or device addressed in the document; or c) The person or a member of the person's household, has a reasonable potential for financial, professional or other personal gain or loss as a result of the issues/content addressed in the document.AATS indicates American Association for Thoracic Surgery; ACC, American College of Cardiology; AHA, American Heart Association; AIG, Association of International Governors; ASE, American Society of Echocardiography; DSMB, Data and Safety Monitoring Board; MRI, magnetic resonance imaging; NIH, National Institutes of Health; NYU, New York University; PARTNERS, Placement Of Aortic Transcatheter Valves; PI, Principal Investigator; SCA, Society of Cardiovascular Anesthesiologists; SCAI, Society for Cardiovascular Angiography and Interventions; STS, Society of Thoracic Surgeons; and VA, Veterans Affairs.
Parasites use polymorphic gene families to evade the immune system or interact with the host. Assessing the diversity and expression of such gene families in pathogens can inform on the repertoire or ...host interaction phenotypes of clinical relevance. However, obtaining the sequences and quantifying their expression is a challenge. In Plasmodium falciparum, the highly polymorphic var genes encode the major virulence protein, PfEMP1, which bind a range of human receptors through varying combinations of DBL and CIDR domains. Here we present a tool, Varia, to predict near full-length gene sequences and domain compositions of query genes from database genes sharing short sequence tags. Varia generates output through two complementary pipelines. Varia_VIP returns all putative gene sequences and domain compositions of the query gene from any partial sequence provided, thereby enabling experimental validation of specific genes of interest and detailed assessment of their putative domain structure. Varia_GEM accommodates rapid profiling of var gene expression in complex patient samples from DBLα expression sequence tags (EST), by computing a sample overall transcript profile stratified by PfEMP1 domain types.
Varia_VIP was tested querying sequence tags from all DBL domain types using different search criteria. On average 92% of query tags had one or more 99% identical database hits, resulting in the full-length query gene sequence being identified (> 99% identical DNA > 80% of query gene) among the five most prominent database hits, for ~ 33% of the query genes. Optimized Varia_GEM settings allowed correct prediction of > 90% of domains placed among the four most N-terminal domains, including the DBLα domain, and > 70% of C-terminal domains. With this accuracy, N-terminal domains could be predicted for > 80% of queries, whereas prediction rates of C-terminal domains dropped with the distance from the DBLα from 70 to 40%.
Prediction of var sequence and domain composition is possible from short sequence tags. Varia can be used to guide experimental validation of PfEMP1 sequences of interest and conduct high-throughput analysis of var type expression in patient samples.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface ...proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7-72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly ...hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative 'slender' to transmissible 'stumpy' bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.
Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly ...improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.
We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.
We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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