Multiplex dispensation order generation for pyrosequencing

This paper introduces the multiplex dispensation order generation problem, a real-life combinatorial problem that arises in the context of analyzing large numbers of short to medium length DNA sequences. The problem is modeled as a constraint optimization problem (COP). We present the COP, its constraint programming formulation, and a custom search procedure. We give some experimental data supporting our design decisions. One of the lessons learnt from this study is that the ease with which the relevant constraints are expressed can be a crucial factor in making design decisions in the COP model

Pyrosequencing™ : A one-step method for high resolution HLA typing

While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing

Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: Comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing

Background: Single-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns.Results: Unexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance.Conclusion: Our data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases. © 2014 Rajatileka et al.; licensee BioMed Central Ltd

CPLOP: The Cal Poly Library of Pyroprints

To date, microbial source tracking (MST), i.e. determining the source of microbial contamination based on the specific strains observed in the environment, often uses methods that are time-consuming, expensive and not always reliable. The biology department at Cal Poly, San Luis Obispo has developed a new method for MST called pyroprinting. Pyroprints can be used as DNA fingerprints for identifying sources of fecal contamination and studying bacterial populations in host animals. The MST method consists of two parts: the pyroprinting process and a database of pyroprints. The Cal Poly Library of Pyroprints (CPLOP) was developed to provide a database application for automating the storage and analysis of pyroprints. The initial version of CPLOP provided support for storing and organizing data related to microbes and their sources. This thesis describes the additional analytical tools needed to fully support pyroprinting as an MST method. This includes the ability to organize pyroprints into dataets, detect erroneous pyroprint data, run analyses to find similarities between bacterial isolates, and cluster isolates into bacterial strains

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.Siirretty Doriast

Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts

The strength and timing of the mitochondrial bottleneck in salmon suggests a conserved mechanism in vertebrates

In most species mitochondrial DNA (mtDNA) is inherited maternally in an apparently clonal fashion, although how this is achieved remains uncertain. Population genetic studies show not only that individuals can harbor more than one type of mtDNA (heteroplasmy) but that heteroplasmy is common and widespread across a diversity of taxa. Females harboring a mixture of mtDNAs may transmit varying proportions of each mtDNA type (haplotype) to their offspring. However, mtDNA variants are also observed to segregate rapidly between generations despite the high mtDNA copy number in the oocyte, which suggests a genetic bottleneck acts during mtDNA transmission. Understanding the size and timing of this bottleneck is important for interpreting population genetic relationships and for predicting the inheritance of mtDNA based disease, but despite its importance the underlying mechanisms remain unclear. Empirical studies, restricted to mice, have shown that the mtDNA bottleneck could act either at embryogenesis, oogenesis or both. To investigate whether the size and timing of the mitochondrial bottleneck is conserved between distant vertebrates, we measured the genetic variance in mtDNA heteroplasmy at three developmental stages (female, ova and fry) in chinook salmon and applied a new mathematical model to estimate the number of segregating units (N(e)) of the mitochondrial bottleneck between each stage. Using these data we estimate values for mtDNA Ne of 88.3 for oogenesis, and 80.3 for embryogenesis. Our results confirm the presence of a mitochondrial bottleneck in fish, and show that segregation of mtDNA variation is effectively complete by the end of oogenesis. Considering the extensive differences in reproductive physiology between fish and mammals, our results suggest the mechanism underlying the mtDNA bottleneck is conserved in these distant vertebrates both in terms of it magnitude and timing. This finding may lead to improvements in our understanding of mitochondrial disorders and population interpretations using mtDNA data

Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses

A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing

BACKGROUND:Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. OBJECTIVE:Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. METHODS:We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. RESULTS:We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. CONCLUSION:The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing