Target enrichment using parallel nanoliter quantitative PCR amplification

Background: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. Results: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. Conclusions: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform’s promise as a targeted resequencing method for multi-gene routine sequencing diagnostics

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities

Background The β-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. Results We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. Conclusion The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study

BACKGROUND: Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. RESULTS: The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. CONCLUSIONS: Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens

Investigating the role of DNA methyltransferases in the mantled somaclonal variation of oil palm

DNA methyltransferase (DMTases) genes have emerged as targets of interest in the exploration of epigenetic mechanisms underlying the mantled variant phenotype in oil palm. Indeed, reduction in global DNA methylation rates and perturbations of floral phenotype could be due to underexpresion of these genes. Therefore, our research efforts focused on the isolation of members of all three families of DNA-methyltranferases identified in higher plants, namely MET, CMT (chromomethylase) and DRM (domain-rearranged). Using both a library screening and a PCR-based approach involving degenerated primers, we successfully cloned partial cDNA sequences belonging to all three families. The respective full-length cDNAs were subsequently obtained through the RACE (Rapid Amplification of cDNA Ends) method. For each DMTase type, we then determined the size of the family in the oil palm genome, and we examined the overall transcription level of each family member individually. The comparative transcription pattern of each class of DMTase was studied in normal and variant tissues through semi-quantitative Reverse-Transcription-PCR (sqRT-PCR) and Real Time quantitative PCR. Our work shows for the first time the characterization of the three DNA methyltransferase gene families in oil palm, and addresses the hypothesis of their role in the determinism of the mantled variant phenotype. (Résumé d'auteur

Differential expression of cellulose synthase (CesA) gene transcripts in potato as revealed by QRT-PCR

Two transgenic potato lines, csr2–1 and csr4–8 that contained two different antisense cellulose synthase (CesA) genes, csr2 and csr4, respectively were crossed. The aim, amongst others, was to investigate the possibility of generating double transformants to validate a hypothetical presence of the proteins of the two CesA genes in the same cellulose synthase enzyme complex. SYBR-Green quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assays were carried out on four CesA gene transcripts (CesA1, 2, 3, and 4) in the wild type genetic background, and on the two antisense CesA gene transcripts (CesA2 and 4) in the progeny resulting from the cross between the two transgenic potato lines. The quantitative RT-PCR analyses revealed different expression patterns of the two CesA genes. The CesA2 mRNA was shown to be relatively more abundant than CesA4 mRNA, regardless of the genetic background, suggesting that the two proteins are not present in the same enzyme complex

MITOCHONDRIAL DNA POLYMORPHISMS AND FERTILITY IN BEEF CATTLE

Two regions of mitochondrial DNA, D-loop and ND-5 were characterized using polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) involving 422 beef cattle of Hereford and composite breeds from Wokalup’s research station. ANOVA models (model I, II) were used to estimate associations between molecular haplotypes and quantitative traits. The phenotypic data used were records on calving rate, defined as the mean number of live calves born over four years, while the genotypic data used were the result of PCR-RFLP analysis in both regions of mitochondrial DNA using 7 restriction enzymes. The results of the present study have provided evidence that mitochondrial polymorphisms in the D-loop and ND-5 regions are associated significantly with fertility. This is the first report of a correlation between mitochondrial polymorphism in D-loop and ND-5 on fertility in beef cattle. Key words: PCR-RFLP, bovine mitochondrial DNA, D-loop, ND-5