scholarly journals A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

2019 ◽  
Author(s):  
Abdul Wahab ◽  
Ayaz Shaukat ◽  
Qasim Ali ◽  
Mubashir Hussain ◽  
Taj Ali Khan ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Disease Transmission ◽  
Diagnostic Performance ◽  
Detection Threshold ◽  
Plasmodium Species ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Diagnostic Tools ◽  
Sequencing Method

AbstractVarious PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput ‘haemoprotobiome’ metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

scholarly journals A high throughput deep amplicon sequencing method to show the emergence and spread of Calicophoron daubneyi in United Kingdom cattle herds

2019 ◽  
Author(s):  
Neil D. Sargison ◽  
Kashif Shahzad ◽  
Stella Mazeri ◽  
Umer Chaudhry
Keyword(s):  
United Kingdom ◽  
Anthelmintic Resistance ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Animal Management ◽  
The United Kingdom ◽  
Sequencing Method ◽  
Cattle Herds ◽  
The Impact ◽  
Cox 1

AbstractThe prevalence of C. daubneyi infection in the United Kingdom has increased, but despite the potential for rumen flukes to cause production loss in ruminant livestock, understanding of their emergence and spread is poor. Here we describe the development of a method to explore the multiplicity of C. daubneyi infection and patterns of the parasite’s emergence and spread, based on Illumina MiSeq deep sequencing of meta barcoded amplicons of a fragment of the mt-COX-1 locus. Our results show high levels of genetic diversity per infection and between populations of 10 to 47 of adult C. daubneyi, each from a total of 32 finished prime cattle consigned to slaughter from northern United Kingdom; with 18 unique mt-COX-1 haplotypes. This has implications for the adaptability of environmental and intermediate host stages of the parasite to changing climatic and animal management conditions, or of parasitic stages to exposure to anthelmintic drugs; potentially allowing for greater pathogenicity, or the development of anthelmintic resistance, respectively. Our results illustrate the impact of high levels of animal movements in the United Kingdom, whereby multiple common mt-COX-1 haplotypes were identified in 26 populations in the absence of geographical clustering of clades.

scholarly journals Molecular epidemiological surveillance of Africa and Asia imported malaria in Wuhan, Central China: comparison of diagnostic tools during 2011–2018

2020 ◽  
Author(s):  
Yiting Xie ◽  
Kai Wu ◽  
Weijia Cheng ◽  
Tingting Jiang ◽  
Yi Yao ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Plasmodium Species ◽  
Public Health Problem ◽  
Imported Malaria ◽  
Central China ◽  
Taqman Probe ◽  
Epidemiological Surveillance ◽  
Diagnostic Tools ◽  
Small Scale ◽  
Blood Samples

Abstract Background Malaria remains a serious public health problem globally. Along with indigenous malaria elimination in China, imported malaria gradually became a major hazard. Well-timed and accurate diagnosis could support immediate therapeutic schedule, reveal the prevalence of imported malaria and avoid the disease transmission. Method Blood samples were collected in Wuhan, China from August 2011 to December 2018. All patients first accepted microscopy and RDT examination. Subsequently, each of the positive or suspected positive cases was engaged in total four human Plasmodium species amplication by using 18S rRNA based nested PCR and Taqman probe based real-time PCR. Then performance of microscopy and two molecular diagnosis methods were analyzed. Importation origin was traced by country and prevalence of Plasmodium species was revealed by year. Results Total 296 blood samples containing 288 microscopy and RDT positive, 7 RDT P. falciparum positive and 1 suspected cases were collected and reanalyzed. After two molecular methods and sequencing detection, 291 cases including 245 P. falciparum , 15 P. vivax , 20 P. ovale , 6 P. malariae and 5 mixed infection (3 P. falciparum + P. ovale , 2 P. vivax + P. ovale ) were confirmed. These patients returned from Africa (95.53%) and Asia (4.47%). Although the prevalence displayed a small-scale fluctuation, the overall trend of the imported cases increased yearly. Conclusions Results emphasized the necessity of combined utilization of the four tools for malaria diagnosis in clinic and field survey around potential risk regions worldwide including Wuhan.

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 Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

2019 ◽  
Author(s):  
Umer Chaudhry ◽  
Qasim Ali ◽  
Imran Rashid ◽  
Muhammad Zubair Shabbir ◽  
Muhammad Abbas ◽  
...  
Keyword(s):  
Species Composition ◽  
Species Interactions ◽  
Control Strategies ◽  
Detection Threshold ◽  
Amplicon Sequencing ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Parasitic Infections ◽  
Sequencing Platform ◽  
Field Samples

AbstractPiroplasmosis is caused by tick-borne haemoprotozoa of the generaTheileriaandBabesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities.TheileriaandBabesiavary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a “haemoprotobiome”. First, four phenotypically-verified species ofTheileriaandBabesiawere used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The “haemoprotobiome” concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

scholarly journals High-Throughput Illumina MiSeq Amplicon Sequencing of Yeast Communities Associated With Indigenous Dairy Products From Republics of Benin and Niger

2019 ◽  
Vol 10 ◽  
Author(s):  
Philippe Sessou ◽  
Santosh Keisam ◽  
Ngangyola Tuikhar ◽  
Mariama Gagara ◽  
Souaïbou Farougou ◽  
...  
Keyword(s):  
High Throughput ◽  
Dairy Products ◽  
Illumina Miseq ◽  
Amplicon Sequencing

Brief history of high-throughput nucleic acid sequencing methods.

Biomics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 27-46
Author(s):  
V.V. Zubov ◽  
D.A. Chemeris ◽  
R.G. Vasilov ◽  
V.E. Kurochkin ◽  
Ya.I. Alekseev
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Chain Growth ◽  
Single Dna Molecules ◽  
Sequencing Method ◽  
Semiconductor Sequencing ◽  
History Of ◽  
High Throughput Dna Sequencing ◽  
Dna Chain ◽  
Chain Polymerization

The processes occurring during the enzymatic growth of the DNA chain in the form of elongation of the molecules, the release of pyrophosphate, proton, thermal energy, and an increase in electrical impedance, which are used in various methods of high-throughput DNA sequencing by synthesis, are briefly considered. The detection of DNA chain growth is controlled by high-voltage gel electrophoresis and has limited scalability. As for mentioned above other by-products of DNA chain polymerization, their detection can be easily scalable, which has led to the emergence of methods for whole genome sequencing of new generations of DNA, which have received the widely used abbreviation NGS - Next Generation Sequencing. However, the attribution of any new sequencing method to a particular generation is sometimes difficult due to the fact that the principle used in it was born earlier than the other one was implemented, which turned out to be less productive in the end. In addition, it is more important to distinguish the methods of new DNA sequencing into two groups in which the massive parallel sequencing of identical matrices takes place or the sequencing of single DNA molecules takes place and last one have received the designation monomolecular sequencing. In this review, along with the classical Sanger method of DNA sequencing, which is still the "gold standard", pyrosequencing, semiconductor sequencing, thermosequencing, electronic sequencing, fluorescent bridge sequencing and sequencing using nanoballs from the first group, as well as monomolecular methods – tSMS sequencing, SMRT sequencing and nanopore sequencing are considered. Attention is paid to the costs of DNA sequencing and the prospects for its development.

scholarly journals Molecular epidemiological surveillance of Africa and Asia imported malaria in Wuhan, Central China: comparison of diagnostic tools during 2011–2018

2020 ◽  
Author(s):  
Yiting Xie ◽  
Kai Wu ◽  
Weijia Cheng ◽  
Tingting Jiang ◽  
Yi Yao ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Plasmodium Species ◽  
Public Health Problem ◽  
Imported Malaria ◽  
Central China ◽  
Taqman Probe ◽  
Epidemiological Surveillance ◽  
Diagnostic Tools ◽  
Small Scale ◽  
Blood Samples

Abstract Background Malaria remains a serious public health problem globally. Along with indigenous malaria elimination in China, imported malaria gradually became a major hazard. Well-timed and accurate diagnosis could support immediate therapeutic schedule, reveal the prevalence of imported malaria and avoid the disease transmission. Method Blood samples were collected in Wuhan, China from August 2011 to December 2018. All patients first accepted microscopy and RDT examination. Subsequently, each of the positive or suspected positive cases was engaged in total four human Plasmodium species amplication by using 18S rRNA based nested PCR and Taqman probe based real-time PCR. Then performance of microscopy and two molecular diagnosis methods were analyzed. Importation origin was traced by country and prevalence of Plasmodium species was revealed by year. Results Total 296 blood samples containing 288 microscopy and RDT positive, 7 RDT P. falciparum positive and 1 suspected cases were collected and reanalyzed. After two molecular methods and sequencing detection, 291 cases including 245 P. falciparum , 15 P. vivax , 20 P. ovale , 6 P. malariae and 5 mixed infection (3 P. falciparum + P. ovale , 2 P. vivax + P. ovale ) were confirmed. These patients returned from Africa (95.53%) and Asia (4.47%). Although the prevalence displayed a small-scale fluctuation, the overall trend of the imported cases increased yearly. Conclusions Results emphasized the necessity of combined utilization of the four tools for malaria diagnosis in clinic and field survey around potential risk regions worldwide including Wuhan.

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