High throughput automated allele frequency estimation by pyrosequencing

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.     

Viral population analysis and minority-variant detection using short read next-generation sequencing

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.     

River otter population size estimation using noninvasive latrine surveys

Across much of North America, river otter (Lontra canadensis) populations were extirpated or greatly reduced by the early 20th century. More recently, reintroductions have resulted in restored populations and the recommencement of managed trapping. Perhaps the best example of these river otter reintroductions occurred in Missouri, regarded as one of the most successful carnivore recovery programs in history. However, abundance estimates for river otter populations are difficult to obtain and often contentious when used to underpin management activities. We assessed the value of latrine site monitoring as a mechanism for quantifying river otter abundance. Analyses of fecal DNA to identify individual animals may result in an improved population estimate and have been used for a variety of mammal species. We optimized laboratory protocols, redesigned existing microsatellite primers, and calculated genotyping error rates to enhance genotyping success for a large quantity of river otter scat samples. We also developed a method for molecular sexing. We then extracted DNA from 1,421 scat samples and anal sac secretions (anal jelly) collected during latrine site counts along 22–34-km stretches representing 8–77% of 8 rivers in southern Missouri in 2009. Error rates were low for the redesigned microsatellites. We obtained genotypes at 7–10 microsatellite loci for 24% of samples, observing highest success for anal jelly samples (71%) and lowest for fresh samples (collected within 1 day of defecation). We identified 63 otters (41 M, 22 F) in the 8 rivers, ranging from 2 to 14 otters per river. Analyses using program CAPWIRE resulted in population estimates similar to the minimum genotyping estimate. Density estimates averaged 0.24 otters/km. We used linear regression to develop and contrast models predicting population size based on latrine site and scat count indices, which are easily collected in the field. Population size was best predicted by a combination of scats per latrine and latrines per kilometer. Our results provide methodological approaches to guide wildlife managers seeking to initiate similar river otter fecal genotyping studies, as well as to estimate and monitor river otter population sizes. © 2011 The Wildlife Society.     

Early detection of population fragmentation using linkage disequilibrium estimation of effective population size

Population subdivision due to habitat loss and modification, exploitation of wild populations and altered spatial population dynamics is of increasing concern in nature. Detecting population fragmentation is therefore crucial for conservation management. Using computer simulations, we show that a single sample estimator of N _e based on linkage disequilibrium is a highly sensitive and promising indicator of recent population fragmentation and bottlenecks, even with some continued gene flow. For example, fragmentation of a panmictic population of N _e = 1,000 into demes of N _e = 100 can be detected with high probability after a single generation when estimates from this method are compared to prefragmentation estimates, given data for ~20 microsatellite loci in samples of 50 individuals. We consider a range of loci (10–40) and individuals (25–100) typical of current studies of natural populations and show that increasing the number of loci gives nearly the same increase in precision as increasing the number of individuals sampled. We also evaluated effects of incomplete fragmentation and found this N _e-reduction signal is still apparent in the presence of considerable migration (m ~ 0.10–0.25). Single-sample genetic estimates of N _e thus show considerable promise for early detection of population fragmentation and decline.     

Blastocystis: subtyping isolates using pyrosequencing technology

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.     

High-throughput species identification of enterococci using pyrosequencing

Here we report the development and validation of an automated high-throughput pyrosequencing-based method for the reliable identification of isolated Enterococcus species. This method exploits the discrete species-specificity of hypervariable groES-EL spacer region and utilizes a universal dispensation order optimized for a wide range of Enterococcus species.     

Assessment of soil fungal communities using pyrosequencing

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.     

Improved performance of pyrosequencing using single-stranded DNA-binding protein

In modern biology, there is a critical need to develop a high-throughput and inexpensive platform for DNA sequencing. Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. In these studies, single-stranded DNA-binding protein (SSB) was added to the primed DNA template prior to the Pyrosequencing reaction. The addition of SSB to a Pyrosequencing reaction system resulted in a read length of more than 30 nucleotides. Improvements were observed as: (i) increased efficiency of the enzymes, (ii) reduced mispriming, as measured by nonspecific signals, (iii) an increase in signal intensity during the reaction, (iv) higher accuracy in reading the number of identical adjacent nucleotides in difficult templates, and (v) longer reads. The usefulness of these results for future Pyrosequencing applications is discussed.     

Hyperparameter estimation using stochastic approximation with application to population pharmacokinetics

A stochastic approximation algorithm is proposed for recursive estimation of the hyperparameters characterizing, in a population, the probability density function of the parameters of a statistical model. For a given population model defined by a parametric model of a biological process, an error model, and a class of densities on the set of the individual parameters, this algorithm provides a sequence of estimates from a sequence of individuals' observation vectors. Convergence conditions are verified for a class of population models including usual pharmacokinetic applications. This method is implemented for estimation of pharmacokinetic population parameters from drug multiple-dosing data. Its estimation capabilities are evaluated and compared to a classical method in population pharmacokinetics, the first-order method (NONMEM), on simulated data.     

Viral population dynamics and virulence thresholds

Viral factors and host barriers influence virally induced disease, and asymptomatic versus symptomatic infection is governed by a 'virulence threshold'. Understanding modulation of virulence thresholds could lend insight into disease outcome and aid in rational therapeutic and vaccine design. RNA viruses are an excellent system to study virulence thresholds in the context of quasispecies population dynamics. RNA viruses have high error frequencies and our understanding of viral population dynamics has been shaped by quasispecies evolutionary theory. In turn, research using RNA viruses as replicons with short generation times and high mutation rates has been an invaluable tool to test models of quasispecies theory. The challenge and new frontier of RNA virus population dynamics research is to combine multiple theoretical models and experimental data to describe viral population behavior as it changes, moving within and between hosts, to predict disease and pathogen emergence. Several excellent studies have begun to undertake this challenge using novel approaches.     

Molecular haplotype determination using allele-specific PCR and pyrosequencing technology

Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3' end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.     

De novo quantitative bisulfite sequencing using the pyrosequencing technology

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.     

A label-free assay of exonuclease activity using a pyrosequencing technique

Enzymes with 3′-5′ exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3′-5′ exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3′ end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.     

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.     

Changes in microbiota population during fermentation of narezushi as revealed by pyrosequencing analysis

Modern Japanese sushi is derived from an archetype, narezushi, which is made by the fermentation of salted fish with rice. Several studies have demonstrated that lactic acid bacteria are dominantly present in narezushi, but no studies have addressed how microbial composition changes during fermentation. In this study, we examined changes in the microbial population in aji (horse mackerel)-narezushi during fermentation by pyrosequencing the 16S ribosomal RNA gene (rDNA). Ribosomal Database Project Classifier analysis revealed that among the 53 genera present, the Lactobacillus population drastically increased during fermentation, while the populations of other bacteria remained unchanged. Basic Local Alignment Search Tool analysis revealed that L. plantarum and L. brevis were the major species. Comparison with other fermented food microbiota indicated high product-dependency of the bacterial composition, which might have been due to the starter-free fermentation process.     

Multi-stage sampling for population estimation

Summary Multi-stage sampling is a convenient technique suited to the desnity estimation of biological populations living in habitats with complicated structures. This paper describes a general method of its application to population estimation in which the preliminary information on the spatial distribution pattern of the population under study can be incorporated as the parameters of the mean crowding-mean relationship. The formulae that are necessary to perform sequential or double sampling plans for its efficient application are derived. The procedure of application of the method is explained with a numerical example. This study was supported by science research fund from the Ministry of Education.