scholarly journals Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuka Torii ◽  
Kazuhiro Horiba ◽  
Satoshi Hayano ◽  
Taichi Kato ◽  
Takako Suzuki ◽  
...  
Keyword(s):  
Kawasaki Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Rna Virus ◽  
Systemic Vasculitis ◽  
Human Herpesvirus ◽  
Acute Stage ◽  
Serum Samples

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

Abstract 132: Methylprednisolone Pulse Therapy for Kawasaki Disease with Symptomatic Myocarditis

Circulation ◽  
2015 ◽  
Vol 131 (suppl_2) ◽  
Author(s):  
yeo hyang kim ◽  
Chae Ok Shin ◽  
Myung Chul Hyun ◽  
Dong Seok Lee
Keyword(s):  
Kawasaki Disease ◽  
Systolic Function ◽  
Systemic Vasculitis ◽  
Cervical Lymphadenopathy ◽  
Febrile Illness ◽  
Pulse Therapy ◽  
Left Ventricular ◽  
Acute Stage ◽  
Z Score ◽  
Intravenous Methylprednisolone

Purpose: Kawasaki disease (KD) is an acute febrile illness of infants and young children that is characterized by a systemic vasculitis, especially involving the coronary arteries. Although, sometimes, subclinical myocarditis is combined in KD, symptomatic myocarditis is extremely uncommon. We report a 7 year old boy who developed hypotension and decreased left ventricular systolic function (EF 40%) in the acute phase of KD. Case: A 7 year old boy (height 115 cm, body weight 20 kg) was admitted because of 2 days of persistent fever and left cervical lymphadenopathy (white blood cell count 17,870 /mm 3 , C reactive protein 23.6 mg/dL). Conjunctiva injection and lip redness developed on the 4th day of illness, and hypotension and tachycardia (SBP 59/DBP 29 mmHg, HR 153/bpm) were combined. The echocardiography revealed a decreased ejection fraction (EF) (40%) without chamber dilatation and normal coronary artery size (LM 1.9mm, z score=-1.3, RCA 2.3mm, z score=0.4). The level of N terminal pro BNP was 28,000 pg/mL. With a diagnosis of KD with myocarditis, he was initially treated with inotropics and intravenous immunoglobulin (2 g/kg). Without clinical improvement in spite of initial treatment, A change of coronary arterial size (LM 2.9mm, z score=1.2, RCA 3.1mm, z score=2.3) was developed and decreased LV systolic function (EF 45%) and fever were persisted. Then, he was given 3 daily pulses of intravenous methylprednisolone followed by tapering doses of oral prednisolone. He showed prompt clinical recovery after pulse therapy of intravenous methylprednisolone (SBP 95/DBP 49 mmHg, HR 98/bpm). Although EF was improved (59%), coronary arterial dilatation was progressed (LM 3.4mm, z score=2.4 RCA 5.5mm, z score=7.9). Conclusions: The present case serves to highlight the fact that methylprednisolone should be considered as the priority in children with KD who have symptomatic myocarditis during the acute stage.

scholarly journals A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

2017 ◽  
Author(s):  
Gregory L. Owens ◽  
Marco Todesco ◽  
Emily B. M. Drummond ◽  
Sam Yeaman ◽  
Loren H. Rieseberg
Keyword(s):  
Allele Frequency ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Molecular Ecology ◽  
Whole Genome Shotgun ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Illumina Hiseq Platform ◽  
Post Hoc

AbstractHigh throughput sequencing using the Illumina HiSeq platform is a pervasive and critical molecular ecology resource, and has provided the data underlying many recent advances. A recent study has suggested that ‘index switching’, where reads are misattributed to the wrong sample, may be higher in new versions of the HiSeq platform. This has the potential to invalidate both published and in-progress work across the field. Here, we test for evidence of index switching in an exemplar whole genome shotgun dataset sequenced on both the Illumina HiSeq 2500, which should not have the problem, and the Illumina HiSeq X, which may. We leverage unbalanced heterozygotes, which may be produced by index switching, and ask whether the under-sequenced allele is more likely to be found in other samples in the same lane than expected based on the allele frequency. Although we validate the sensitivity of this method using simulations, we find that neither the HiSeq 2500 nor the HiSeq X have evidence of index switching. This suggests that, thankfully, index switching may not be a ubiquitous problem in HiSeq X sequence data. Lastly, we provide scripts for applying our method so that index switching can be tested for in other datasets.

scholarly journals Bracoviruses recruit host integrases for their integration into caterpillar’s genome

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009751
Author(s):  
Zehua Wang ◽  
Xiqian Ye ◽  
Yuenan Zhou ◽  
Xiaotong Wu ◽  
Rongmin Hu ◽  
...  
Keyword(s):  
High Throughput ◽  
Plutella Xylostella ◽  
Late Stage ◽  
High Throughput Sequencing ◽  
Early Stage ◽  
Parasitic Wasp ◽  
Host Genome ◽  
Sequencing Analysis ◽  
Dna Viruses ◽  
Cotesia Vestalis

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential “8+5” nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar’s genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.

scholarly journals High-throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating variants

2021 ◽  
Author(s):  
Rafaela S. Fontenele ◽  
Simona Kraberger ◽  
James Hadfield ◽  
Erin M. Driver ◽  
Devin Bowes ◽  
...  
Keyword(s):  
Population Structure ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Genomic Diversity ◽  
Contact Tracing ◽  
Public Health Response ◽  
Genetic Population ◽  
Genomic Epidemiology ◽  
Derived Data

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from a zoonotic spill-over event and has led to a global pandemic. The public health response has been predominantly informed by surveillance of symptomatic individuals and contact tracing, with quarantine, and other preventive measures have then been applied to mitigate further spread. Non-traditional methods of surveillance such as genomic epidemiology and wastewater-based epidemiology (WBE) have also been leveraged during this pandemic. Genomic epidemiology uses high-throughput sequencing of SARS-CoV-2 genomes to inform local and international transmission events, as well as the diversity of circulating variants. WBE uses wastewater to analyse community spread, as it is known that SARS-CoV-2 is shed through bodily excretions. Since both symptomatic and asymptomatic individuals contribute to wastewater inputs, we hypothesized that the resultant pooled sample of population-wide excreta can provide a more comprehensive picture of SARS-CoV-2 genomic diversity circulating in a community than clinical testing and sequencing alone. In this study, we analysed 91 wastewater samples from 11 states in the USA, where the majority of samples represent Maricopa County, Arizona (USA). With the objective of assessing the viral diversity at a population scale, we undertook a single-nucleotide variant (SNV) analysis on data from 52 samples with >90% SARS-CoV-2 genome coverage of sequence reads, and compared these SNVs with those detected in genomes sequenced from clinical patients. We identified 7973 SNVs, of which 5680 were “novel” SNVs that had not yet been identified in the global clinical-derived data as of 17th June 2020 (the day after our last wastewater sampling date). However, between 17th of June 2020 and 20th November 2020, almost half of the SNVs have since been detected in clinical-derived data. Using the combination of SNVs present in each sample, we identified the more probable lineages present in that sample and compared them to lineages observed in North America prior to our sampling dates. The wastewater-derived SARS-CoV-2 sequence data indicates there were more lineages circulating across the sampled communities than represented in the clinical-derived data. Principal coordinate analyses identified patterns in population structure based on genetic variation within the sequenced samples, with clear trends associated with increased diversity likely due to a higher number of infected individuals relative to the sampling dates. We demonstrate that genetic correlation analysis combined with SNVs analysis using wastewater sampling can provide a comprehensive snapshot of the SARS-CoV-2 genetic population structure circulating within a community, which might not be observed if relying solely on clinical cases.

scholarly journals Target enrichment of long open reading frames and ultraconserved elements to link microevolution and macroevolution in non-model organisms

2021 ◽  
Author(s):  
Claudia Ortiz-Sepulveda ◽  
Mathieu Genete ◽  
Christelle Blassiau ◽  
Cécile Godé ◽  
Christian Albrecht ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Recent Common Ancestor ◽  
Ultraconserved Elements ◽  
Most Recent Common Ancestor ◽  
Reading Frames

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced a substantial dataset. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites), although some branches lack support in the latter case. Variant calling on the exome of Coelaturini from the Malawi Basin produced ~2,000 SNPs per population pair. Nucleotide diversity and population differentiation was low compared to previous estimates in mollusks, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming non-specific sequence data obtained for Coelaturini of the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays an identical gene order to that of the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for the development of integrative genomic studies on micro- and macroevolutionary dynamics in non-model organisms.

scholarly journals Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

2018 ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

AbstractAmplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.ImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

scholarly journals GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

2018 ◽  
Author(s):  
Devika Ganesamoorthy ◽  
Minh Duc Cao ◽  
Tania Duarte ◽  
Wenhan Chen ◽  
Lachlan Coin
Keyword(s):  
High Throughput ◽  
Tandem Repeat ◽  
Copy Number ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Complex Diseases ◽  
Sequencing Analysis ◽  
Reference Dataset ◽  
Long Read

ABSTRACTBackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations.MethodsWe report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68% and 83% for capture sequence data and 200X WGS data respectively, improving to 87% and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25%, 14%, 12% and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

scholarly journals GrigoraSNPs: Optimized HTS DNA Forensic SNP Analysis

2017 ◽  
Author(s):  
Darrell O. Ricke ◽  
Anna Shcherbina ◽  
Adam Michaleas ◽  
Philip Fremont-Smith
Keyword(s):  
High Throughput ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Sequence Data ◽  
Snp Analysis ◽  
Analysis Pipeline ◽  
Sequencing Technologies ◽  
High Throughput Dna Sequencing

AbstractHigh throughput DNA sequencing technologies enable improved characterization of forensic DNA samples enabling greater insights into DNA contributor(s). Current DNA forensics techniques rely upon allele sizing of short tandem repeats by capillary electrophoresis. High throughput sequencing enables forensic sample characterizations for large numbers of single nucleotide polymorphism loci. The slowest computational component of the DNA forensics analysis pipeline is the characterization of raw sequence data. This paper optimizes the SNP calling module of the DNA analysis pipeline with runtime results that scale linearly with the number of HTS sequences (patent pending)[1]. GrigoraSNPs can analyze 100 million reads in less than 5 minutes using 3 threads on a 4.0 GHz Intel i7-6700K laptop CPU.

Sign in / Sign up

Export Citation Format

Share Document