Fragment DNA Sequencing Research Articles

PASTMUS: mapping functional elements at single amino acid resolution in human cells

Identification of functional elements for a protein of interest is important for achieving a mechanistic understanding. However, it remains cumbersome to assess each and every amino acid of a given protein in relevance to its functional significance. Here, we report a strategy, PArsing fragmented DNA Sequences from CRISPR Tiling MUtagenesis Screening (PASTMUS), which provides a streamlined workflow and a bioinformatics pipeline to identify critical amino acids of proteins in their native biological contexts. Using this approach, we map six proteins—three bacterial toxin receptors and three cancer drug targets, and acquire their corresponding functional maps at amino acid resolution.

Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics.

The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

Soymilk bio-enrichment by indigenously isolated riboflavin-producing strains of Lactobacillus plantarum

Lactobacilli (n = 68) isolated from human feces and fermented milk products were screened for the production of riboflavin (vitamin B2) by culturing into riboflavin assay medium (RAM). Cell-free culture supernatants from positive isolates (BBC32A, BBC32B, BBC33 and BIF43) were transferred onto RAM agar pre-seeded with Enterococcus faecalis MTCC2729 (a riboflavin auxotroph). The enhanced growth of B2-auxotroph confirmed the bioavailability of produced vitamin. Isolate BBC32B produced the highest riboflavin (319 ± 36 μg/l), followed by BBC33 (304 ± 91 μg/l), BBC32A (276 ± 8 μg/l) and BIF43 (257 ± 91 μg/l). All four isolates contained riboflavin genes ribG, ribB, ribA and ribH. Sequencing of DNA fragments amplified from the 16S–23S rRNA intergenic spacer region and areas flanking the 23S rRNA gene grouped these isolates within the species Lactobacillus plantarum. Identifications were confirmed by sequencing 1300-bp of amplified 16S rDNA fragments. Fermentation of soymilk by single cultures of L. plantarum BBC32B, BBC33 and BIF43 yielded 49.05%, 38.97% and 35.94% riboflavin enrichment respectively, which is more than 18.75% recorded in literature for Lactobacillus fermentum MTCC8711. Maximum levels of riboflavin were obtained within 12 h of fermentation in soymilk. Lactobacillus plantarum BBC32B may be used as starter culture for developing of riboflavin-enriched soymilk.

Integrative taxonomy reveals first country record of Hyalinobatrachium mondolfii Señaris and Ayarzagüena 2001, and distribution range extensions forCochranella nola Harvey 1996, and Rulyrana spiculata Duellman 1976 (Anura: Centrolenidae) in Peru.

We used an integrative taxonomy approach to investigate the taxonomic identity of several populations of glassfrogs from Peru, which are notoriously challenging to identify due to their overall similarity in morphology and coloration. We relied on comparisons of morphology, bioacoustics, and partial fragments of 16S rRNA DNA sequences. We report for the first time the presence of Hyalinobatrachium mondolfii in Peru, being this the southernmost locality known for the species. Likewise, we update and extend the distribution ranges of Rulyrana spiculata and Cochranella nola in the Andes of Peru, provide a 16S sequence of a topotype of R. spiculata, and confirm its presence in Bolivia. For all three species, we increase the current knowledge on their geographic distribution and genetic and phenotypic variation.

Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

Combination of Fetal Fraction Estimators Based on Fragment Lengths and Fragment Counts in Non-Invasive Prenatal Testing.

The reliability of non-invasive prenatal testing is highly dependent on accurate estimation of fetal fraction. Several methods have been proposed up to date, utilizing different attributes of analyzed genomic material, for example length and genomic location of sequenced DNA fragments. These two sources of information are relatively unrelated, but so far, there have been no published attempts to combine them to get an improved predictor. We collected 2454 single euploid male fetus samples from women undergoing NIPT testing. Fetal fractions were calculated using several proposed predictors and the state-of-the-art SeqFF method. Predictions were compared with the reference Y-based method. We demonstrate that prediction based on length of sequenced DNA fragments may achieve nearly the same precision as the state-of-the-art methods based on their genomic locations. We also show that combination of several sample attributes leads to a predictor that has superior prediction accuracy over any single approach. Finally, appropriate weighting of samples in the training process may achieve higher accuracy for samples with low fetal fraction and so allow more reliability for subsequent testing for genomic aberrations. We propose several improvements in fetal fraction estimation with a special focus on the samples most prone to wrong conclusion.

Abstract 2671: Omics unveils a specific signature of tumor dormancy in two murine models of leukemia and melanoma

Abstract The goal of the project was to integrate “omics” data, including genomic, epigenetic, transcriptomic and proteomic profiles, to identify a specific signature of tumor dormancy. Materials and Methods: Two murine models of tumor dormancy, B16F1 malignant melanoma and DA1-3b myeloid leukemia, were generated and exome sequencing of dormant cells and parental cells was performed.Mutations were identified. Epigenetic alterations were detected by ChIP sequencing. Briefly, repressed and activated genes were identified by sequencing DNA fragments obtained after immunoprecipitation with antibodies against H3K9 and H3K27 and against histone H3K4me3, respectively. DNA Methylation analysis was assessed by Methylation Specific PCR (MSP). Transcriptomic analysis was conducted using Agilent arrays. Proteomes were compared by mass spectrometry. For the calcium homeostasis analysis, time courses of cytosolic Ca2+ concentration were measured using the ratiometric dye Fura-2. Results: Exome analysis identified a specific gene signature of tumor dormancy in each model. Previously characterized mutations in human melanoma and myeloid leukemia were found in the murine models of B16F1 melanoma (e.g., Pten, Brca2, and CKit) and myeloid leukemia (e.g., Flt3 and Dnmt3b), thus reinforcing the relevance of these models for translational research on human pathology. ChIP sequencing revealed a specific epigenetic signature of dormant cells compared to parental cells. Global proteomic analysis showed a deregulation of proteins involved in many metabolic pathways in both models. Despite the absence of common gene mutations in the two models tested, several genes involved in calcium homeostasis were found to be mutated (Transient Receptor Potential, Store Operated calcium channels ). Regarding their epigenetic regulation assessed by MSP, no difference was observed in methylation level. Nevertheless, functional assays showed profound alterations in calcium homeostasis in the two dormant cell lines compared to the parental cell lines, suggesting a functional role of one or several receptors or ion channels in calcium signaling in tumor dormancy. Conclusions: Taken together, “omics” analyses indicate that the regulation of tumor cell dormancy is extremely complex. Therefore, an integrated approach is crucial to fully understand tumor dormancy. Bioinformatics and integrative analyses will provide a better understanding of mechanisms controlling dormancy. Finally, functional analysis of calcium homeostasis revealed a common calcium signature in dormant tumor cells compared to parental cells. The genes involved in this altered intracellular calcium flux are currently being elucidated. Citation Format: Yasmine Touil, Loïc Lemonnier, Pascaline Segard, Martin Figeac, Frédéric Leprêtre, Audrey Vincent, Lucile Noyer, Maxence Wisztorski, Jean-Pascal Gimeno, Jean-Pascal Meneboo, Guillemette Marot, Isabelle Fournier, Thierry Idziorek, Bruno Quesnel. Omics unveils a specific signature of tumor dormancy in two murine models of leukemia and melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2671.

Abstract 2572: Integrated viral sequences in esophageal cancer tissue

Abstract The human genome exhibits a significant degree of invasion by viral genomes. During the course of evolution, several RNA and DNA viruses have become integrated into the vertebrate genome and there is no reason to believe that the process has ended. Integrated viral DNA may account for as much as 10% of the human genome and it is very likely that they may act as carcinogens by altering normal cellular gene expression. In this study we have compared the viral DNA sequences present in normal and tumor tissues from esophageal squamous cell carcinoma (ESCC) samples. DNA was extracted from paired normal and tumor biopsies and subjected to whole genome sequencing. Raw data run folders were converted to FASTQ files and progressed through a QC pipeline. Normal and tumor data sets were then blasted and aligned against an extensive viral sequence database using specialized DRAGEN software, so that all integrated viral DNA fragment sequences were identified. The identified viral sequences were then compared between the paired normal and tumor samples. Using this approach, a large number of viral DNA sequences were identified; these included human papilloma viruses, Herpes simplex viruses, adenoviruses and Hepatitis C viruses. Furthermore, some unexpected viruses of interest were also identified. These include the Autographa Californica Nucleopolyhedrovirus, the Emiliana Huxleyi virus 86, the Gryllus bimaculatus nudivirus and the Trichodysplasia spinulosa-associated polyomavirus, present in different tumour DNA samples. Amplification and rearrangements of endogenous retroviruses such as the Human Endogenous Retrovirus K113 were also observed. Endogenous retroviruses (ERV) can serve as long-lasting viral reservoirs and provide evidence for the coexistence between retroviruses and their hosts over many generations. Defective ERVs can also recombine to affect the expression of adjacent genes. It has been proposed that integrated viral sequences may play a possible role in the etiology and promotion of diseases such as cancer, thus targeted studies of specific viral sequences may elucidate the mechanisms of gene disruption and may ultimately lead to the development of more effective therapeutic interventions. Note: This abstract was not presented at the meeting. Citation Format: Victoria A. Patten, Lamech Mwaphaga, Christopher G. Mathew, Denver T. Hendricks, M. Iqbal Parker. Integrated viral sequences in esophageal cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2572.

A set of 156 SNP markers for teak (Tectona grandis Linn. f.)

Novel single nucleotide polymorphism loci were identified for teak (Tectona grandis Linn. f.) using a combined Next Generation Sequencing approach on the Illumina MiSeq system with genotyping on the Agena MassARRAY iPLEX™ platform. Three hundred and forty four putative loci were identified through sequencing of DNA fragments following a modified double digest Restriction Associated DNA Sequencing method. One hundred and eighty nine loci successfully amplified in 172 individuals from 20 populations in Myanmar. A total of 33 loci were removed from the final panel as two showed evidence of deviation from Hardy–Weinberg equilibrium, 17 were significantly linked and 23 were non-polymorphic. For the remaining 156 loci, expected heterozygosity ranged from 0.041 to 0.456 and FST from 0.062 to 0.632. These genetic resources will prove useful for future studies into the population genetics and phylogeography of this important and iconic timber species.

Cost‐efficient high throughput capture of museum arthropod specimen DNA using PCR‐generated baits

Abstract Gathering genetic data for rare species is one of the biggest remaining obstacles in modern phylogenetics, particularly for megadiverse groups such as arthropods. Next generation sequencing techniques allow for sequencing of short DNA fragments contained in preserved specimens >20 years old, but approaches such as whole‐genome sequencing are often too expensive for projects including many taxa. Several methods of reduced representation sequencing have been proposed that lower the cost of sequencing per specimen, but many remain costly because they involve synthesizing nucleotide probes and target hundreds of loci. These datasets are also frequently unique for each project and thus generally incompatible with other similar datasets. Here, we explore utilization of in‐house generated DNA baits to capture commonly utilized mitochondrial and ribosomal DNA loci from insect museum specimens of various ages and preservation types without the a priori need to know the sequence of the target loci. Both within species and cross‐species capture are explored, on preserved specimens ranging in age from one to 54 years old. We found most samples produced sufficient amounts of data to assemble the nuclear ribosomal rRNA genes and near complete mitochondrial genomes and produce well‐resolved phylogenies in line with expected results. The dataset obtained can be straightforwardly combined with the large cache of existing Sanger‐sequencing‐generated data built up over the past 30 years and targeted loci can be easily modified to those commonly used in different taxa. Furthermore, the protocol we describe allows for inexpensive data generation (as low as ~$35/sample), of at least 20 kilobases per specimen, for specimens at least as old as ~1965, and can be easily conducted in most laboratories. If widely applied, this technique will accelerate the accurate resolution of the Tree of Life especially on non‐model organisms with limited existing genomic resources.

Hb Knossos (HBB: c.82G\u2009>\u2009T), \u03b2-globin CD 5 (\u2212CT) (HBB: c.17_18delCT) and \u03b4-globin CD 59 (\u2212a) (HBD: c.179delA) mutations in a Syrian patient with \u03b2-thalassemia intermedia

BackgroundBeta thalassemia (β-thal) is an inherited hemoglobin disorder characterized by reduced synthesis of the hemoglobin that results in microcytic hypochromic anemia. β-Thalassemia intermedia (TI) is a clinical term of intermediate gravity between the carrier state and β-thalassemia major (β -TM).Case presentationWe describe a 12-year-old male proband originating from Al-Quneitra province - southwest Syria. Hematological investigations revealed, pallor and anemia (Hb 9 g/dl). The mean cell volume (MCV) 64 fL; mean cell hemoglobin (MCH) 21.8 pg. Capillary electrophoresis (CE) electropherogram revealed low level of Hb A1 (36.2%), high level of Hb F (62.2%) and low level of Hb A2 (1.6%). The proband requires blood transfusion occasionally. Direct DNA sequencing and Polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) for mutations detection were used. The molecular analysis revealed the presence of rare β+ Hb Knossos codon 27 (G > T) (HBB: c.82G > T) variant associated with β0 codon 5 [−CT] (HBB: c.17_18delCT) mutation in beta-globin (β-globin) gene and δ0 codon 59 [−A] (HBD: c.179delA) mutation in delta-globin (δ-globin) gene. The proband tested negative for the common deletional forms of alpha thalassemia (α-thal). Polymorphism of the Xmn-I locus (HBG2: c.-211C > T) revealed that the proband had a homozygous [TT] for Xmn-1 locus.ConclusionsTo our knowledge, this is the first report of beta thalassemia intermedia due to combination of Hb Knossos /codon 5 [−CT] associated with δ0 codon 59 [−A] in Syrian patient. On the other hand, in Syria, β-thal carriers who have low level of Hb A2 due to decreased δ-chain production, different δ-thal gene mutations must be screened to avoid the failure diagnosis of β-thal disease.

A fluorescence method for homogeneous detection of influenza A DNA sequence based on guanine-quadruplex-N-methylmesoporphyrin IX complex and assistance-DNA inhibition.

In his study, we report a fluorescence method for homogeneous detection of influenza A (H1N1) DNA sequence based on G-quadruplex-NMM complex and assistance-DNA (A-DNA) inhibition. The quadruplex-based functional DNA (QBF-DNA), composed of a complementary probe to the target H1N1 DNA sequence and G-rich fragment, was designed as the signal DNA. The A-DNA consisted of two parts, one part was complementary to target H1N1 DNA and the other part was complementary to the signal DNA. In the absence of target H1N1 DNA, the G-rich fragment of QBF-DNA can form G-quadruplex-NMM complex, which outputted a fluorescent signal. With the presence of target H1N1 DNA, QBF-DNA, and A-DNA can simultaneously hybridize with target H1N1 DNA to form double-helix structure. In this case, the A-DNA partially hybridized with the QBF-DNA, which inhibited the formation of G-quadruplex-NMM complex, leading to the decrease of fluorescent signal. Under the optimum conditions, the fluorescence intensity was inversely proportional to the concentration of target H1N1 DNA over the range from 25 to 700 pmol/L with a detection limit of 8 pmol/L. In addition, the method is target specific and practicability, and would become a new diagnostic assay for H1N1 DNA sequence and other infectious diseases.

Parallel Object Compositions for the Search of Sequences DNA Strings in the Construction of Gnomes

Within an environment of parallel objects, an approach of structured parallel programming and the paradigm of the orientation to objects show a programming method based on high level parallel compositions or HLPCs to solve two problems of combinatorial optimization: grouping fragments of DNA sequences and the parallel exhaustive search (PES) of RNA strings that help the sequence and the assembly of DNAs. The pipeline and farm models are shown as HLPCs under the object orientation paradigm and with them it is proposed the creation of a new HLPCs that combines and uses the previous ones to solve the cited problems. Each HLPC proposal contains a set of predefined synchronization constraints between processes, as well as the use of synchronous, asynchronous and asynchronous future modes of communication. This article shows the algorithms that solve the problems, their design and implementation as HLPCs and the performance metrics in their parallel execution using multicores and video accelerator card.

Zoological origin survey of commercial hippocampus in Chinese herbal markets by morphological and DNA sequencing identification

Hippocampus is a precious animal medicine in Chinese herbal medicines. Numerous seahorse species possessing similar morphology were used as commercial hippocampus in herbal markets. Clarifing the zoological of commercial hippocampus in herbal markets is a crucial issue, which contributed to establish authentication and quality control standard. This study investigated 1 156 dried seahorse samples collected from eight main herbal markets using CO Ⅰ fragment DNA sequencing coupling with morphological identification. The results showed that 23 seahorse species were present in the China TCM market. Among them, five species were officially listed in China Pharmacopoeia, seven species namely winged seahorse (Hippocampus alatus), giraffe seahorse (H. camelopardalis), knysna seahorse (H. capensis), beibuwan seahorse (H. casscsio), half-spiny seahorse (H. semispinosus), Europe seahorse (H. hippocampus), zebra seahorse (H. zebra) were found in herbal markets for the first time. The present DNA sequences analysis coupling with morphological identification method could also use to survey the species origin of other Chinese herbal medicines in herbal markets.

Porous Zero-Mode Waveguides for Picogram-Level DNA Capture.

We have recently shown that nanopore zero-mode waveguides are effective tools for capturing picogram levels of long DNA fragments for single-molecule DNA sequencing. Despite these key advantages, the manufacturing of large arrays is not practical due to the need for serial nanopore fabrication. To overcome this challenge, we have developed an approach for the wafer-scale fabrication of waveguide arrays on low-cost porous membranes, which are deposited using molecular-layer deposition. The membrane at each waveguide base contains a network of serpentine pores that allows for efficient electrophoretic DNA capture at picogram levels while eliminating the need for prohibitive serial pore milling. Here, we show that the loading efficiency of these porous waveguides is up to 2 orders of magnitude greater than their nanopore predecessors. This new device facilitates the scaling-up of the process, greatly reducing the cost and effort of manufacturing. Furthermore, the porous zero-mode waveguides can be used for applications that benefit from low-input single-molecule real-time sequencing.

From Cellular Diversity to the Roots of Variability

From Cellular Diversity to the Roots of Variability

Toxoplasma gondii infection in stranded St. Lawrence Estuary beluga Delphinapterus leucas in Quebec, Canada.

The St. Lawrence Estuary (SLE) beluga Delphinapterus leucas in Quebec, Canada, is endangered due to intensive hunting in the 19th and 20th centuries and subsequent anthropogenic contamination and human activities in the region. Infectious disease is a primary cause of death in this population. The protozoan parasite Toxoplasma gondii is reported in numerous marine mammal species, including beluga. In the present study, 55 tissue samples (heart and brain) collected from 34 stranded SLE beluga were analysed by PCR followed by DNA sequencing and restriction fragment length polymorphism analysis (RFLP) to determine the PCR prevalence and genotypes of T. gondii in these beluga. Of 34 beluga tested, 44% were positive for T. gondii by PCR, with males having a higher prevalence of infection than females and with more infected neonates and juveniles than adults. Molecular analyses indicated that all T. gondii infecting stranded SLE beluga grouped into genotype II, which predominates in humans. While our results indicate that a high prevalence of stranded beluga are PCR-positive for T. gondii infection, very few deaths are attributed to toxoplasmosis based on published necropsy results. Toxoplasma gondii can cause a range of diseases, including neurological deficits, and more data are needed to investigate this parasite's effect on population recovery.

Introduction to the Special Issue on Next Generation Sequencing: Short General Overview of NGS

he publication of the double helix DNA structure in 1953 [1] was the kick-off of numerous efforts to understand and unravel the complexity of the human genome. It took 50 years until the human genome project, based on Sanger sequencing, was completed in 2003 [2]. This was followed by what may be considered a revolution in the field of DNA analysis: the introduction of Next Generation Sequencing (NGS) in 2005 consisting of massively parallel sequencing of DNA fragments which allows the high throughput analysis of large numbers of genes or of the whole genome [3].

Detection of Phytoplasmas Associated with Tomato Witches’ Broom and Big Bud Disease in Egypt

Symptoms of witches’ broom were observed on tomato plants in some fields in Aswan governorate, Egypt. Molecular investigation was done with phytoplasma-detection primer P1/P7 to amplify a fragment of the 16S rRNA gene plus spacer region and beginning of 23Sr gene. RFLP analyses and sequencing of DNA fragments amplified by R16F2n/R2 primers indicated that these phytoplasmas are closely related and could be classified in the 16SrII group. Phylogenetic analyses of 41 accessions of 16S ribosomal RNA gene sequences of ‘Candidatus phytoplasmas’ comprising the strain from Egypt and representative strains from GenBank confirmed that the phytoplasma from tomato cluster with other strains all classified in subgroup 16SrII-D. Therefore, it could be useful to use the RFLP methodology in rapid and specific screenings of this phytoplasma presence in Egyptian tomato plants

Helicobacter pylori 23S rRNA gene mutations associated with clarithromycin resistance in chronic gastritis in Vietnam.

Data about the prevalence of the A2142C, A2142G, and A2143G mutations in 23S rRNA gene is still limited. The aim of this study was to determine the prevalence of these mutations in 23S rRNA gene of H. pylori vietnamese strains. One hundred and sixty-nine patients with H. pylori-positive chronic gastritis were examined. H. pylori was detected by rapid urease test and Polymerase chain reaction (PCR). Total DNA was extracted from gastric biopsy specimens. A2142C, A2142G, and A2143G mutations were detected by DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP). A2143G mutation was detected in 36.1% of samples, A2142G mutation in 3.6%, while A2142C mutation was not found in any case. The mixture of wild-type and mutation strains was found in 50% of specimens with A2142G, in 23% of specimens with A2143G mutation. There was no association of 23S rRNA gene point mutations with gender or age. However, an association between the heterogeneity of mutation and age was evidenced, with mean age of the group of pure A2143G higher than the group of wild-type/A2143G mixture, and rate of the wild-type/A2143G mixture higher in patients under 40 years of age. A2143G mutation was prominent, while A2142C mutation was not found in the 23S rRNA gene. PCR-RFLP has revealed a reliable assay allowing a rapid and cost-effective detection of clarithromycin-resistant strains. This is useful in countries as Vietnam with high prevalence of clarithromycin-resistance before choosing optimal therapy for H. pylori eradication.