High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 585-594 ◽  
Author(s):  
ARTHUR KOCHER ◽  
SOPHIE VALIÈRE ◽  
ANNE-LAURE BAÑULS ◽  
JÉRÔME MURIENNE
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Genomic Region ◽  
Kinetoplast Dna ◽  
Sequence Comparisons ◽  
Conserved Sequence ◽  
Species Complexes ◽  
Pcr Products ◽  
Minicircle Sequence

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

scholarly journals New Arsenate Reductase Gene (arrA) PCR Primers for Diversity Assessment and Quantification in Environmental Samples

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Babur S. Mirza ◽  
Darwin L. Sorensen ◽  
R. Ryan Dupont ◽  
Joan E. McLean
Keyword(s):  
Drinking Water ◽  
Human Health ◽  
High Throughput ◽  
In Silico ◽  
High Throughput Sequencing ◽  
Gene Diversity ◽  
Pcr Primers ◽  
Potential Threat ◽  
Groundwater Samples ◽  
Reductase Gene

ABSTRACT The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (≥90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (AsIII) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of AsV to AsIII. In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (AsIII) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.

scholarly journals PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e116264 ◽  
Author(s):  
Florian Barbi ◽  
Claudia Bragalini ◽  
Laurent Vallon ◽  
Elsa Prudent ◽  
Audrey Dubost ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Lignocellulolytic Enzymes ◽  
Genes Encoding ◽  
Fungal Genes

Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes

1989 ◽  
Vol 9 (3) ◽  
pp. 1365-1367
Author(s):  
D S Ray
Keyword(s):  
Base Pair ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Kinetoplast Dna ◽  
Base Pairs ◽  
Conserved Sequence ◽  
Diverse Species ◽  
Pair Sequence ◽  
Sequence Region ◽  
Minicircle Sequence

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.

scholarly journals Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes.

1989 ◽  
Vol 9 (3) ◽  
pp. 1365-1367 ◽  
Author(s):  
D S Ray
Keyword(s):  
Base Pair ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Kinetoplast Dna ◽  
Base Pairs ◽  
Conserved Sequence ◽  
Diverse Species ◽  
Pair Sequence ◽  
Sequence Region ◽  
Minicircle Sequence

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.

scholarly journals High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 684 ◽  
Author(s):  
Toufic Elbeaino ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Elisa Trioano ◽  
Thierry Candresse ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Small Scale ◽  
Rt Pcr ◽  
Cyclamen Persicum ◽  
Sequence Variations ◽  
Pcr Products ◽  
Complete Sequences ◽  
Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

scholarly journals A Nested-PCR-Based Schizodeme Method for Identifying Leishmania Kinetoplast Minicircle Classes Directly from Clinical Samples and Its Application to the Study of the Epidemiology of Leishmania tropica in Pakistan

1998 ◽  
Vol 36 (10) ◽  
pp. 2877-2881 ◽  
Author(s):  
Harry A. Noyes ◽  
Hugh Reyburn ◽  
J. Wendy Bailey ◽  
David Smith
Keyword(s):  
Nested Pcr ◽  
Clinical Laboratory ◽  
Variable Region ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Leishmania Tropica ◽  
Guanidine Thiocyanate ◽  
Kinetoplast Dna ◽  
Detection And Identification ◽  
Pcr Products

A nested PCR was developed to amplify the variable region of the kinetoplast minicircles of all Leishmania species which infect mammals. Each Leishmania parasite contains approximately 10,000 kinetoplast DNA minicircles, which are unequally distributed among approximately 10 minicircle classes. The PCR primers were designed to bind within the 120-bp conserved region which is common to all minicircle classes; the remaining approximately 600 bp of each minicircle is highly conserved within each minicircle class but highly divergent between classes. The nested PCR generated a strong signal from a minimum of 0.1 fg of Leishmania DNA. Restriction digests of the amplicons from the highest dilutions suggested that minicircles from only a limited number of minicircle classes had acted as template in the reaction. One PCR product was directly sequenced and found to be derived from only one minicircle class. Since the primers amplify all minicircle classes, this indicated that as little as 1/10 of one Leishmania parasite was present in the PCR template. This demonstrated that the nested PCR achieved very nearly the maximum theoretically possible sensitivity and is therefore a potentially useful method for diagnosis. The nested PCR was tested for sensitivity on 20 samples from patients from the Timargara refugee camp, Pakistan. Samples were collected by scraping out a small amount of tissue with a scalpel from an incision at the edge of the lesion; the tissue was smeared on one microscope slide and placed in a tube of 4 M guanidine thiocyanate, in which the sample was stable for at least 1 month. DNA for PCR was prepared by being bound to silica in the presence of 6 M guanidine thiocyanate; washed in guanidine thiocyanate, ethanol, and acetone; and eluted with 10 mM Tris-HCl. PCR products of the size expected for Leishmania tropica were obtained from 15 of the 20 samples in at least one of three replicate reactions. The negative samples were from lesions that had been treated with glucantime or were over 6 months old, in which parasites are frequently scanty. This test is now in routine use for the detection and identification of Leishmaniaparasites in our clinical laboratory. Fingerprints produced by restriction digests of the PCR products were defined as complex or simple. There were no reproducible differences between the complex restriction patterns of the kinetoplast DNA of any of the parasites from Timargara camp with HaeIII and HpaII. The simple fingerprints were very variable and were interpreted as being the product of PCR on a limited subset of minicircle classes, and consequently, it was thought that the variation was determined by the particular minicircle classes that had been represented in the template. The homogeneity of the complex fingerprints suggests that the present epidemic of cutaneous leishmaniasis in Timargara camp may be due to the spread of a single clone of L. tropica.

scholarly journals The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing

PLoS ONE ◽  
2007 ◽  
Vol 2 (2) ◽  
pp. e197 ◽  
Author(s):  
Jonas Binladen ◽  
M. Thomas P. Gilbert ◽  
Jonathan P. Bollback ◽  
Frank Panitz ◽  
Christian Bendixen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Parallel Sequencing

scholarly journals Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA

2011 ◽  
Vol 39 (1) ◽  
pp. 321-326 ◽  
Author(s):  
Tobias Klatte ◽  
Laura Evans ◽  
Rebekah N. Whitehead ◽  
Jeffrey A. Cole
Keyword(s):  
Nitrate Reductase ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Periplasmic Nitrate Reductase ◽  
Pcr Products ◽  
Successful Design ◽  
The One ◽  
Bacterial Genes ◽  
Biological Nitrogen

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.

scholarly journals CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Dave J. Baker ◽  
Alp Aydin ◽  
Thanh Le-Viet ◽  
Gemma L. Kay ◽  
Steven Rudder ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rapid Expansion ◽  
Library Preparation ◽  
Method Performance ◽  
Genome Coverage ◽  
Maximum Likelihood Tree ◽  
Pcr Products

AbstractWe present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.

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