Direct Sequencing Data Research Articles

A machine learning algorithm based on multi-omics biomarkers for the detection of tumor microsatellite instability.

1554 Background: Microsatellite instability (MSI) occurs via defects in mismatch repair (MMR) and is characterized by high tumor mutational burden (TMB) and a robust inflammatory response. Accurate MSI-high classification of immune-evasive tumors is vital due to the effectiveness of immune checkpoint inhibitor (ICI) therapy, such as pembrolizumab, which is FDA approved for all MSI-high tumors irrespective of anatomic origin. Detection methods of MSI-status vary widely, including immunohistochemistry (IHC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). We developed a machine learning (ML) model to predict MSI status in patients with solid tumors using comprehensive genomic and immune profiling (CGIP), independent of direct sequencing data from microsatellite sites. Methods: We analyzed samples from 1,838 patients with colorectal cancer (CRC) by CGIP, which included DNA panel testing (523 genes) for pathogenic single nucleotide variants (pSNV) and determination of TMB (mut/Mb), and RNA sequencing (397 genes) for gene expression (GEX). The Boruta algorithm was used to select key genomic and GEX changes associated with MSI status. A distributed gradient boosting algorithm created a predictive model that was trained on 70% of the cohort. The model was tested on the remaining CRC cohort (Test), and in the PanCancer Atlas CRCs (TCGA), endometrial adenocarcinomas (EMCA), and CRC with indeterminate MSI testing by CGIP. Performance of the trained model was assessed using sensitivity, specificity, and positive (PPV) and negative (NPV) predictive value. Results: Feature selection identified 79 genes with pSNVs, 63 GEX changes, and TMB as informative for MSI prediction. The model showed high predictive accuracy in differentiating MSI-H tumors. Of the 39 cases that failed MSI component of CGIP (Indeterminate), 17 had MMR IHC results available, of which 1 demonstrated loss of MLH1/PMS2 and the other 16 showed intact (normal) expression. Testing of this cohort identified 3 cases as MSI-high, at least one of which was confirmed by IHC. No cases with intact MMR protein IHC were identified as MSI-high by the algorithm. Conclusions: Our ML-driven approach accurately assessed MSI status of CRC and EMCA using CGIP data. This approach also identified potential cases with MSI-high status where direct sequencing of microsatellites failed. This study highlights a method to identify patients with potential MSI-high status for orthogonal screening with the MSI component of a test fails. [Table: see text]

Emerging novel sequence types of Staphylococcus aureus in Pakistan

BackgroundDespite an increasing incidence of Staphylococcus aureus infection and dissemination in Pakistan, the epidemiology of different Staphylococcus aureus research clones has been the subject of only a small number of investigations. By analyzing the collected data sequence, this study was designed to study the epidemiology of Staphylococcus aureus in the area using multilocus sequence typing (MLST). MethodsA total of 1015 staphylococcus strains collected from the city's tertiary care facilities were biochemically screened, followed by antimicrobial susceptibility testing against a panel of 13 antibiotics. Analyzed methicillin-resistant Staphylococcus aureus (MRSA) was subjected to molecular characterization using multilocus sequence typing (MLST), clonal complex analysis, recombination testing, and phylogenetic analysis. ResultsApproximately 421 bacteria were verified as Staphylococcus aureus by biochemical analysis. 57% of the isolates exhibited multidrug resistance, of which 89% were found to be methicillin-resistant Staphylococcus aureus (MRSA). MLST results in a total of 39 sequence types (ST) and 5 clonal complexes (CC), out of which twenty-two STs were newly documented worldwide. The most common CC identified was CC8. The direct sequencing data also revealed significant shifts at MLST loci, with point mutations resulting in the aroE-343 and tpi-278 alleles. ConclusionsThis study concludes that there is high diversity in the locally circulating clones of Staphylococcus aureus present in nature and that they are defined by their geographic epidemiology. These findings have practical implications for public health, including the need for tailored infection control strategies, antibiotic stewardship, global surveillance, and a deeper understanding of bacterial evolution.

Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform.

A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant isvery important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developedmethod is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked withdifferent percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λex/λem of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.

Diagnostically significant single nucleotide polymorphisms in the <I>env</I>-gene locus for SNP-genotyping of <I>Bovine leukemia virus</I>

Relevance. The etiological agent in the development of persistent lymphocytosis and lymphosarcoma in cattle is Bovine leukemia virus. Phylogenetic analysis of sequenced nucleotide sequences of the BLV env-gene locus is the main, but not the only, approach to pathogen genotypic classification.Metods. The aim of the study was to identify single-nucleotide polymorphisms in the locus of the Bovine leukemia virus env-gene, considered as diagnostically significant for SNP genotyping of BLV based on the analysis of direct sequencing data of the PCR-product.Results. Alignment of the nucleotide sequences of the env-gene locus of 101 BLV type isolates of known Bovine leukemia virus genotypes revealed 155 single nucleotide polymorphisms, of which 35 SNP were considered diagnostically significant. At the same time, for BLV SNP-genotyping, an analysis of 22 characterized single nucleotide polymorphisms is sufficient, 14 of which are genotype-specific in relation to nine genotypes of the studied viral pathogen (C232G for the 3rd, G84A for the 4th, A117G, T326C and C412T — 5th, C394T — 8th, C40G — 9th, C132T — 10th, T113A and T256C — the 11th, T49C and G140A — the 12th, C83A and T304C — the 13th genotypes, thereby ensuring their identification. The other 8 characterized polymorphic positions are not genotype-specific, but the interpretation of the results of their detection has identification value in relation to the remaining 4 BLV genotypes: 1st (G205G and A337A), 2nd (T220C and A334G), 6th (A127R, C132C, T153C and C341C) and 7th (T220C and A334A) respectively.

Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores.

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies. We introduce the key concepts of MoP2 and provide a step-by-step fully reproducible and complete example of how to use the workflow for the analysis of S. cerevisiae total RNA samples sequenced using MinION flowcells. The workflow starts with the pre-processing of raw FAST5 files, which includes basecalling, read quality control, demultiplexing, filtering, mapping, estimation of per-gene/transcript abundances, and transcriptome assembly, with support of the GPU computing for the basecalling and read demultiplexing steps. The secondary analyses of the workflow focus on the estimation of RNA poly(A) tail lengths and the identification of RNA modifications. The MoP2 code is available at https://github.com/biocorecrg/MOP2 and is distributed under the MIT license.

LIQA: long-read isoform quantification and analysis

Long-read RNA sequencing (RNA-seq) technologies can sequence full-length transcripts, facilitating the exploration of isoform-specific gene expression over short-read RNA-seq. We present LIQA to quantify isoform expression and detect differential alternative splicing (DAS) events using long-read direct mRNA sequencing or cDNA sequencing data. LIQA incorporates base pair quality score and isoform-specific read length information in a survival model to assign different weights across reads, and uses an expectation-maximization algorithm for parameter estimation. We apply LIQA to long-read RNA-seq data from the Universal Human Reference, acute myeloid leukemia, and esophageal squamous epithelial cells and demonstrate its high accuracy in profiling alternative splicing events.

LongGF: computational algorithm and software tool for fast and accurate detection of gene fusions by long-read transcriptome sequencing

BackgroundLong-read RNA-Seq techniques can generate reads that encompass a large proportion or the entire mRNA/cDNA molecules, so they are expected to address inherited limitations of short-read RNA-Seq techniques that typically generate < 150 bp reads. However, there is a general lack of software tools for gene fusion detection from long-read RNA-seq data, which takes into account the high basecalling error rates and the presence of alignment errors.ResultsIn this study, we developed a fast computational tool, LongGF, to efficiently detect candidate gene fusions from long-read RNA-seq data, including cDNA sequencing data and direct mRNA sequencing data. We evaluated LongGF on tens of simulated long-read RNA-seq datasets, and demonstrated its superior performance in gene fusion detection. We also tested LongGF on a Nanopore direct mRNA sequencing dataset and a PacBio sequencing dataset generated on a mixture of 10 cancer cell lines, and found that LongGF achieved better performance to detect known gene fusions over existing computational tools. Furthermore, we tested LongGF on a Nanopore cDNA sequencing dataset on acute myeloid leukemia, and pinpointed the exact location of a translocation (previously known in cytogenetic resolution) in base resolution, which was further validated by Sanger sequencing.ConclusionsIn summary, LongGF will greatly facilitate the discovery of candidate gene fusion events from long-read RNA-Seq data, especially in cancer samples. LongGF is implemented in C++ and is available at https://github.com/WGLab/LongGF.

Whole-genome sequencing of Mycobacterium tuberculosis directly from clinical samples for high-resolution genomic epidemiology and drug resistance surveillance: an observational study

Direct whole-genome sequencing of Mycobacterium tuberculosis from clinical specimens will be a major breakthrough in tuberculosis diagnosis and control. To date, direct whole-genome sequencing has never been used in genomic epidemiology, and its accuracy in transmission inference remains unknown. We investigated the technical challenges imposed by direct whole-genome sequencing, and used it to infer transmission clusters and predict drug resistance. Using an optimised workflow, we did direct whole-genome sequencing for 37 clinical specimens from 23 tuberculosis patients. Nine sputum samples from nine patients who were infected with different non-tuberculous mycobacteria and culture-negative for tuberculosis were used as controls in the qPCR assays and pre-sequencing runs. Additionally, 780 clinical isolates in the region of Comunidad Valenciana (Spain) were whole-genome sequenced between Jan 1, 2014, and Dec 31, 2016. We analysed the genomic variants to build a tuberculosis transmission network for the region, including the clinical specimens, and to predict drug susceptibility profiles. After sequencing 37 clinical specimens, 28 specimens (22 [85%] of 26 smear-positive and six [55%] of 11 smear-negative) met the quality criteria for downstream analysis. All 28 clinical specimens clustered with their matching culture isolates, with a median distance of 0 single nucleotide polymorphisms. Of the 28 clinical specimens, 16 (57%) were accurately assigned to ten transmission clusters in the region, and 12 (43%) were unique cases. Transmission inferences and drug-susceptibility predictions from direct whole-genome sequencing data were concordant with sequences from corresponding cultures and phenotypic drug-susceptibility testing. Complete genomic analysis, within a week of specimen receipt, cost €217 per sample (excluding personnel costs). Direct whole-genome sequencing could be used to accurately delineate transmission clusters of tuberculosis and conduct culture-independent surveillance. Compared with conventional approaches, direct whole-genome sequencing allows researchers to do real-time genomic epidemiology and drug resistance surveillance in settings where culture and drug susceptibility testing are not available. European Research Council; Ministerio de Ciencia, Innovación y Universidades (Spanish Government).

Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer.

Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate‐limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

Performance of probe polymerization-conjunction-agarose gel electrophoresis in the rapid detection of KRAS gene mutation.

This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. The genotype of mutation sites was determined based on the occurrence of target bands in the corresponding lanes of the reaction tubes through polymerization-conjunction of the probes, probe purification and amplification, and agarose gel electrophoresis. Circulating DNA samples were obtained from the plasma of 72 patients with lung cancer, which were identified based on six mutation sites (G12S, G12R, G12C, G12D, G12A, and G12V) of codon 12 of the KRAS gene. The detection results were compared with direct sequencing data. The proposed detection method is characterized by simple operation, high specificity, and high sensitivity (2%). This method can detect the mutations of three samples at G12S, G12R, and G12A. In the direct sequencing spectra of these samples, the genotype could not be determined due to the lack of evident sequencing peaks that correspond to the basic group of mutations. In conclusion, a simple and rapid method was established based on probe polymerization-conjunction-agarose gel electrophoresis for detecting KRAS gene mutations. This method can be applied to the conventional mutation detection of inhomogeneous samples.

Metagenomics workflow analysis of endophytic bacteria from oil palm fruits

Next-Generation Sequencing (NGS) has become a powerful sequencing tool for microbial study especially to lead the establishment of the field area of metagenomics. This study described a workflow to analyze metagenomics data of a Sequence Read Archive (SRA) file under accession ERP004286 deposited by University of Sao Paulo. It was a direct sequencing data generated by 454 pyrosequencing platform originated from oil palm fruits endophytic bacteria which were cultured using oil-palm enriched medium. This workflow used SortMeRNA to split ribosomal reads sequence, Newbler (GS Assembler and GS Mapper) to assemble and map reads into genome reference, BLAST package to identify and annotate contigs sequence, and QualiMap for statistical analysis. Eight bacterial species were identified in this study. Enterobacter cloacae was the most abundant species followed by Citrobacter koseri, Seratia marcescens, Latococcus lactis subsp. lactis, Klebsiella pneumoniae, Citrobacter amalonaticus, Achromobacter xylosoxidans, and Pseudomonas sp. respectively. All of these species have been reported as endophyte bacteria in various plant species and each has potential as plant growth promoting bacteria or another application in agricultural industries.

ITS2, a Better DNA Barcode than ITS in Identification of Species in Artemisia L.

ObjectiveTo select a more suitable DNA barcode to identify the species in Artemisia L. MethodsITS, ITS2, and three other major universal barcode candidates (matK, rbcL, and psbA-trnH) were evaluated in the identification efficiency using a total of 1433 sequences downloaded from GenBank representing 343 species in Artemisia L. ITS and ITS2 were evaluated in the PCR and sequencing rate, sequencing peak quality (Q value), and misread rate. One hundred and twelve A. annua samples were collected from 11 populations across over China, which were amplified with universal primers on the ITS and ITS2 regions. ResultsITS and ITS2 shared a higher identification efficiency and exhibited 71.43% and 64.11% detectability at the species level, respectively. The Q values of ITS and ITS2 showed that the direct PCR sequencing data were reliable for the ITS2 region and ITS exhibited poor sequencing trace quality. In certain sites, the ITS sequences exhibited reading ambiguities and errors, indicating that the misread and deletion sites in the ITS region would incorrectly inflate the identification ratio. ConclusionITS2 is a suitable barcode for identification of species in Artemisia L., which enlarges the optimal range of divergence levels for taxonomic inferences using ITS2 sequences.

Abstract P3-07-11: High activation of PI3K pathway defined by PIK3CA mutation, PTEN, and INPP4B expression are associated with trastuzumab efficacy in HER2-positive breast cancer

Abstract Background Aberrations of phosphoinositide-3-kinase (PI3K) pathway are extensively found in many human cancers through several mechanisms, including mutation or amplification of PIK3CA and loss of phosphatase and tensin homolog (PTEN) and inositol polyphosphate 4-phosphatase-II(INPP4B). In breast cancer, a number of studies have suggested the putative mechanism of resistance to trastuzumab therapy in terms of PI3K pathway activation. We aimed to evaluate the predictive relevance of these biomarkers to trastuzumab efficacy in HER2-positive disease. Patients and Methods A total of 43 breast cancer patients with HER2-positive who received both neoadjuvant treatment and surgery at Kumamoto University Hospital between 2004 and 2012 were selected. The regimens of chemotherapy included anthracycline or taxane-containing drugs in combination with trastuzumab. Using pretreatment tumor tissues, PIK3CA mutations (E542K, E545K, and H1047R) were analyzed by direct dideoxynucleotide sequencing and digital PCR methods. Additionally, the expressions of PTEN, pAkt, and INPP4B were assessed by immunohistochemistry (IHC). Results The overall pathological complete response (pCR) rate was 60%. Direct sequencing detected PIK3CA mutations in 21% of all patients, whereas digital PCR detected them in 26 % when the cutoff point of the mutation was set at 1%. In some cases, it was difficult to differentiate mutant DNA from artifact by direct sequencing, but we could identify the mutation clearly using digital PCR. We found the correlation between the proportion of the PIK3CA mutation and the pCR rate; the pCR rates in the patients with PIK3CA mutations with cut-off of 1%, 10% and 20% were 55%, 29%, and 0%, respectively. There were no significant correlations of clinicopathological features with PIK3CA mutations, copy number status, PTEN, and pAkt expression. Low INPP4B expression was associated with larger tumor size (P = 0.035), and higher nuclear grade (P = 0.031) compared to high expression. We evaluated the contribution of biomarkers related to the PI3K pathway to the prediction of pCR by logistic regression models. In multivariate analysis, activation of the PI3K pathway due to either PIK3CA mutation or low PTEN expression were related to poorer response to trastuzumab (OR of predictive pCR was 0.11, P = 0.041). Similarly, high activation defined as PIK3CA mutation or low expression of PTEN or INPP4B tend to have lower pCR (OR was 0.14, P = 0.064). Conclusions 1. Digital PCR has potential to complement the direct sequencing data, leading to more accurate measurement of the mutation frequency. 2. Our findings provide additional support for the recently published studies regarding activating mutation in PIK3CA in HER2-positive breast cancer, and further suggest that integrated biomarkers of PIK3CA mutation, PTEN, INPP4B are stronger predictors of trastuzumab response than either one alone. Citation Format: Sueta A, Yamamoto Y, Takeshita T, Yamamoto-Ibusuki M, Iwase H. High activation of PI3K pathway defined by PIK3CA mutation, PTEN, and INPP4B expression are associated with trastuzumab efficacy in HER2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-11.

Molecular phylogeny of the Chinese weedy rice based on nrDNA ITS and two cpDNA sequences

Direct sequencing data from the nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) region and two chloroplast DNA (cpDNA) fragments (psbA-trnH and petGtrnP) of Chinese weedy rice (Oryza stavia L. f. sp. spontanea) were analyzed separately and simultaneously in the MEGA 6.0 software. The nrDNA, cpDNA and cpDNA+nrDNA phylogenetic trees showed that the weedy rice collected from Northern China, Oryza sativa Japonica group and Oryza rufipogon, were on the one phylogenic node whereas weedy rice were collected from Southern China and Oryza sativa Indica group were on the other phylogenic node. The results revealed that there were geographical and indica-japonica differentiations in Chinese weedy rice which were corresponding to their geographical distribution and cultivated rice (Oryza sativa) indica-japonica differentiation. These results demonstrated that Chinese weedy rice from different regions have diverse origins and they were the relative origin group of the cultivated rice as there is no wild form available in Northern China. Additionally, clone sequence variation of the nrDNA ITS regions in intraindividual weedy rice provides evidence origin of other Oryza taxa through the process of hybridization or introgression. Data from this research further confirmed that the Chinese weedy rice was evolved from local rice varieties (as the female parent) which naturally hybridized with other oryza materials.

Development of a multiplex and cost-effective genotype test toward more personalized medicine for the antiplatelet drug clopidogrel.

There has been a wide range of inter-individual variations in platelet responses to clopidogrel. The variations in response to clopidogrel can be driven by genetic polymorphisms involved in the pathway of absorption, distribution, metabolism, excretion, and the target receptor P2Y12. A set of genetic variants known for causing variations in clopidogrel responses was selected, which included CYP2C19*2, *3, *17, CYP2B6*4, *6, *9, CYP3A4*18, CYP3A5*3, MDR1 2677G > T/A, 3435C > T, and P2Y12 H2 (742T > C). The simultaneous detection of these 10 variants was developed by using a multiplex PCR and single-base extension (MSSE) methodology. The newly developed genotyping test was confirmed by direct DNA sequencing in the representative positive control samples and validated in an extended set of 100 healthy Korean subjects. Genotyping results from the developed MSSE exhibited a perfect concordance with the direct DNA sequencing data and all of variants tested in 100 healthy Korean subjects were in agreement with Hardy-Weinberg equilibrium (p > 0.05). The present molecular diagnostic studies provide an accurate, convenient, and fast genotyping method for the detection of multiple variants. This would be helpful for researchers, as well as clinicians, to use genetic information toward more personalized medicine of clopidogrel and other antiplatelet drugs in the future.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data.

Establishment of a method for detection of IVS-2-654(C>T) and -28(A>G) gene mutations by high-resolution melting analysis in β-thalassemia and its clinical application

Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application. Methods Two common mutations ［IVS-2-654(C>T) and -28(A>G)］ of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity, sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children′s Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data. Results HRM method could detect the mutations ［IVS-2-654(C>T) and -28(A>G)］ of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types′ Tm were smaller than 0.1%.And minimum 103 copies of DNA of each assay and 10% mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654（C>T）heterozygous mutation and 9-28（A>G）heterozygous mutation and none homozygous mutation. Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established, which is a convenient, rapid, specific, sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.(Chin J Lab Med, 2012，35：730-735) Key words: beta-Thalassemia; Mutation; High resolution melting

Molecular Detection and Partial Characterization of Tomato Yellow Leaf Curl Virus in Sri Lanka

Tomato Yellow Leaf Curl Virus (TYLCV) is an important plant virus on one of the economically most important vegetable crops; tomato (Lycopersicon esculentum Mill.). This had not been molecularly detected before, in Sri Lanka. TYLCV-GN-SL was isolated from apparently infected tomato plants using modified Cetyltrimethyl Ammonium Bromide (CTAB) method in Gannoruwa. Associated Begomoviruses were detected using Deng 541/Deng 540 and AV 494/AC 1048 primer pairs. TYLCV was detected for the first time in tomato in Sri Lanka using P1V/P4C, TYLCV specific primer pair. Nucleotide sequence of coat protein of isolated TYLCV-GN-SL proved that the Indian strain of ToLC virus was closely related to Tomato Leaf Curl Sri Lanka Virus (TLCV-SL: 97%) and Tomato leaf curl Geminivirus (TLCGV: 93%) through direct sequencing data. TLCV-SL was confirmed as TYLCV isolate. TYLCV was molecularly detected from major tomato growing districts like Badulla, Nuwara-Eliya, Kandy and Matale in Sri Lanka.

An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus

Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.

Development of a low‐density DNA microarray for diagnosis of target‐site mutations of pyrethroid and organophosphate resistance mutations in the whitefly Bemisia tabaci

Rapid and accurate detection of mutations related to insecticide resistance is essential for development of resistance management strategies to support sustainable agriculture. The M918V, L925I and T929V mutations of the voltage-gated sodium channel gene (vgsc) and the F392W mutation of the acetylcholinesterase I gene (ace1) are reportedly associated with resistance to pyrethroids and organophosphates, respectively, in Bemisia tabaci. In order to detect known base substitutions in the ace1 and vgsc genes, a low-density microarray with an allele-specific probe was developed. Specific regions of the ace1 and vgsc gene mutations were amplified by multiplex asymmetrical PCR using Cy3-labelled primers, and then the PCR products were hybridised on the microarray. After analysing the probe signal data, the microarray containing 12 allele-specific probes produced a unique pattern of probe signals for field DNA samples of B. tabaci. To determine the optimal cut-off value of each probe, receiver operating characteristic (ROC) curve analysis was conducted using SPSS. Among 60 individual samples, microarray data for 57 samples were consistent with direct sequencing data. Although many molecular detection methods have been employed to monitor insecticide resistance, the present microarray provides rapid and accurate identification of target mutations in B. tabaci for resistance management.