Reference DNA Sequence Research Articles

The first report of Dactylogyrus formosus Kulwiec, 1927 (Monogenea: Dactylogyridae) from goldfish (Carassius auratus) in central China.

Dactylogyrus formosus Kulwiec, 1927 (Monogenea: Dactylogyridae), widely distributed across Eurasia, is considered as one of the most frequently encountered ectoparasites of goldfish (Carassius auratus). In the present study, D. formosus on gills of cultured goldfish was reported in central China for the first time, using the methods of morphological characters and molecular markers. It was characterized by "Anchoratoid-Wegeneri" type anchor with elongate inner and unobvious outer root. The morphology of the opisthaptoral hard parts of D. formosus superficially resembles species of D. arcuatus Yamaguti, 1942 parasitized crucian carp, using light and scanning electron microscopy. However, the anchor total length of D. arcuatus (range 90-102) is almost double the size of D. formosus (range 43.7-57.1) which allows for their rapid discrimination from each other; on the other hand, the two species also can be easily separated by the shape of the male copulatory organ. The morphological description of D. formosus is supplemented with phylogenetic analysis of a reference DNA sequence spanning 18S rDNA and ITS1. What is more, this study also redescribes comprehensively D. formosus, providing for the first time a full ten point-to-point morphometric measurements of the hard parts, and importantly, a photographic record of the armature of the haptor and the male copulatory organ.

A novel locally guided genome reassembling technique using an artificial ant system

DNA reassembling is an NP-hard problem (Brun, Theor Comput Sci 395:31---46, 2008; Medvedev et al 2007; Ma and Lombardi 2008). The present article presents a locally guided global learning system to solve the problem of genome reassembling. We have used a reference DNA sequence which is 99 % similar to an unknown DNA sequence. Two different sequences from the same organism generally have around 99 % similarity (Wei et al 2007). We have considered different DNA sequences from NCBI website (http://www.ncbi.nlm.nih.gov). Then we have simulated the tasks of cloning the sequence, followed by shearing the clones to a number of short reads. In our algorithm, we have introduced a new concept in the task of DNA reassembling using Ant Colony Optimization, where pheromone concentration is proportional to the score of assembled DNA fragments with some known reference sequences within the same organism. Unlike local overlapping, we have used here local alignment score of short reads with some known local reference region as the heuristic information. The result shows that our algorithm is capable of reassembling at par with the state-of-the-art. DNA reassembling techniques may need a massive parallel computation and huge memory space (Kurniawan et al 2008) because of size ~109bp of DNA sequences of mammals (Miller et al, Genomics 95:315---327, 2010; Blazewicz et al, Comput Biol Chem 33:224---230, 2009; Butler et al, Genome Res 18:810---820, 2008; Joshi et al 2011; Stupar et al, Arch Oncol 19:3---4, 2011; Quail et al, BMC Genomics 13:1471---2164, 2012), and ACO is inherently concurrent in nature (Dorigo and Stutzle 2004). Due to lack of appropriate computational resources, we had to confine ourselves to deal with the sequences of length up to ~105bp. We have considered 22 sequences of different organism, including Homo sapiens BRCA1 (127429bp) gene. For large sequences, we have applied hierarchical BAC-by-BAC sequencing (Fig. 2) (Myers, Comput Sci Eng 1:33---43, 1999), to stitch the individual segments to retrieve the original DNA sequence.

An infection of Gyrodactylus kobayashii Hukuda, 1940 (Monogenea) associated with the mortality of goldfish (Carassius auratus) from central China.

Goldfish, Carassius auratus, widely distributed across Eurasia, is one of the earliest fish domesticated for ornamental purposes. A series of diseases break out due to high-density culture of goldfish, causing significant economic losses. Here, we report for the first time an infection of Gyrodactylus kobayashii associated with the high mortality of goldfish from a fish farm in Anziying township of Henan province, China, following their transfer to several aquariums. In the first 2 weeks after goldfish (n = 4200, weight 10.24 ± 1.45 g) transfer to aquariums, a total of 3335 goldfish were lost, representing 79.4 % of the initial stocked biomass. The examination showed that no other pathogens were found in this batch of fishes, except for G. kobayashii. The high burdens of G. kobayashii/fish (264.7, range 100-450) were the primary reason for the high mortality, although this was not the sole cause. In addition, the isolate of G. kobayashii from goldfish (C. auratus) in Henan province of central China was described. The morphological characterization was performed using morphometric measurements and drawings of opisthaptoral hard parts of the parasites. The molecular description was performed based on phylogenetic analysis of a reference DNA sequence spanning 5.8S and ITS-2. Importantly, the present study provides for the first time a full 25 point-to-point morphometric measurements and high-resolution images of attachment organ of G. kobayashii.

A cost-effective straightforward protocol for shotgun Illumina libraries designed to assemble complete mitogenomes from non-model species

The mitogenome is an inescapable tool in conservation biology studies. Yet, its routine sequencing may remain tricky despite next-generation sequencing technologies. An enrichment step is often necessary but not always straightforward depending on the initial DNA quality or quantity. Furthermore, the availability of close mitochondrial DNA reference sequences for non-model species limits the primer design for long-range PCR or bait synthesis. Here we propose an easy and cost-effective protocol without enrichment step for building and sequencing multiplexed Illumina libraries from small quantities of either high-quality or degraded genomic DNA. We validated the approach through the successful assembly of the complete mitogenome of 60 bats and 7 tunicates. Our protocol allows the sequencing and assembly of mitochondrial genomes from non-model species with sufficient coverage for applications in conservation genetics.

Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥0.34, p = <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region.

Sequencing of the interferon regulatory factor 6 (IRF6) gene and correlation to its phenotypes in familial non-syndromic cleft lip and palate in North Indian population

The incidence of orofacial clefting varies depending upon geographical location, ethnicity, sex and even socioeconomic status. The study aimed to test the contribution of interferon regulatory factor 6 (IRF6) gene allelic variation to the incidence of non-syndromic cleft lip and/or cleft palate (CL/P) from the population of North India. Patients from 12 North Indian families with non-syndromic cleft lip with or without cleft palate (NSCL/P) with at least two members affected were clinically evaluated and tested. DNA was extracted from the blood, and three exonic part of the IRF6 gene (exons 2, 5 and 7) were amplified by PCR. Direct sequencing was performed by PCR product, and sequencing results were compared with the reference DNA sequence of the IRF6 gene. Three SNPs (rs861019, rs2013162 and rs2235372) in 4 out of 12 families were observed. SNP rs861019 was detected in family no. 4 (F4) around exon 2, SNP 2013162 was detected in family no. 10 (F10) and SNP 2235372 was detected in family nos. 5 and 9 (F5 and F9). This study demonstrates that the three SNPs within the IRF6 gene are associated with NSCL/P, suggesting IRF6 gene sequence variability and variation in this gene, providing first genetic clue to the NSCL/P phenotype in North Indian families. This is our initial step to demonstrate the involvement of the IRF6 gene in NSCL/P patients in North India. Further extensive study and statistical analysis are needed to validate our result. Level of Evidence: Level IV, diagnostic study.

Race, Time and Folded Objects: The HeLa Error

Given their commitment to practices, science studies have bestowed considerable attention upon objects. We have the boundary object, the standardized package, the network object, the immutable mobile, the fluid object, even a fire object has entered the scene. However, these objects do not provide us with a way of understanding their historicity. They are timeless, motionless pictures rather than things that change over time, and while enacting ‘historical moments’ they do not make visible the histories they contain within them. What kind of object could embody history and make that history visible? Inspired by Michel Serres, I suggest the folded object is a way to attend to the temporality and spatiality of objects. In this article I explore this new object by unravelling the history of a DNA reference sequence. I show how, ever since it was produced in the early 1980s, attempts have been made to filter race out of the sequence. That effort has failed due to what one could call ‘political noise’. Making and remaking the sequence have left traces that cannot be erased.

A tool for mapping Single Nucleotide Polymorphisms using Graphics Processing Units

BackgroundSingle Nucleotide Polymorphism (SNP) genotyping analysis is very susceptible to SNPs chromosomal position errors. As it is known, SNPs mapping data are provided along the SNP arrays without any necessary information to assess in advance their accuracy. Moreover, these mapping data are related to a given build of a genome and need to be updated when a new build is available. As a consequence, researchers often plan to remap SNPs with the aim to obtain more up-to-date SNPs chromosomal positions. In this work, we present G-SNPM a GPU (Graphics Processing Unit) based tool to map SNPs on a genome.MethodsG-SNPM is a tool that maps a short sequence representative of a SNP against a reference DNA sequence in order to find the physical position of the SNP in that sequence. In G-SNPM each SNP is mapped on its related chromosome by means of an automatic three-stage pipeline. In the first stage, G-SNPM uses the GPU-based short-read mapping tool SOAP3-dp to parallel align on a reference chromosome its related sequences representative of a SNP. In the second stage G-SNPM uses another short-read mapping tool to remap the sequences unaligned or ambiguously aligned by SOAP3-dp (in this stage SHRiMP2 is used, which exploits specialized vector computing hardware to speed-up the dynamic programming algorithm of Smith-Waterman). In the last stage, G-SNPM analyzes the alignments obtained by SOAP3-dp and SHRiMP2 to identify the absolute position of each SNP.Results and conclusionsTo assess G-SNPM, we used it to remap the SNPs of some commercial chips. Experimental results shown that G-SNPM has been able to remap without ambiguity almost all SNPs. Based on modern GPUs, G-SNPM provides fast mappings without worsening the accuracy of the results. G-SNPM can be used to deal with specialized Genome Wide Association Studies (GWAS), as well as in annotation tasks that require to update the SNP mapping probes.

A Universal Method for Species Identification of Mammals Utilizing Next Generation Sequencing for the Analysis of DNA Mixtures

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method’s robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.

DNA barcode identification of lichen-forming fungal species in the Rhizoplaca melanophthalma species-complex (Lecanorales, Lecanoraceae), including five new species

Recent studies using sequence data from multiple loci and coalescent-based species delimitation have revealed several species-level lineages within the phenotypically circumscribed taxon Rhizoplaca melanophthalma sensu lato. Here, we formally describe five new species within this group, R. occulta, R. parilis, R. polymorpha, R. porterii, and R. shushanii, using support from the coalescent-based species delimitation method implemented in the program Bayesian Phylogenetics and Phylogeography (BPP) as the diagnostic feature distinguishing new species. We provide a reference DNA sequence database using the ITS marker as a DNA barcode for identifying species within this complex. We also assessed intraspecific genetic distances within the six R. melanophthalma sensu lato species. While intraspecific genetic distances within the five new species were less than or equal to the lowest interspecific pairwise comparison values, an overlap in genetic distances within the R. melanophthalma sensu stricto clade suggests the potential for additional phenotypically cryptic lineages within this broadly distributed lineage. Overall, our results demonstrate the potential for accurately identifying species within the R. melanophthalma group by using molecular-based identification methods.

A highly accurate heuristic algorithm for the haplotype assembly problem

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common form of genetic variation in human DNA. The sequence of SNPs in each of the two copies of a given chromosome in a diploid organism is referred to as a haplotype. Haplotype information has many applications such as gene disease diagnoses, drug design, etc. The haplotype assembly problem is defined as follows: Given a set of fragments sequenced from the two copies of a chromosome of a single individual, and their locations in the chromosome, which can be pre-determined by aligning the fragments to a reference DNA sequence, the goal here is to reconstruct two haplotypes (h1, h2) from the input fragments. Existing algorithms do not work well when the error rate of fragments is high. Here we design an algorithm that can give accurate solutions, even if the error rate of fragments is high.ResultsWe first give a dynamic programming algorithm that can give exact solutions to the haplotype assembly problem. The time complexity of the algorithm is O(n × 2t × t), where n is the number of SNPs, and t is the maximum coverage of a SNP site. The algorithm is slow when t is large. To solve the problem when t is large, we further propose a heuristic algorithm on the basis of the dynamic programming algorithm. Experiments show that our heuristic algorithm can give very accurate solutions.ConclusionsWe have tested our algorithm on a set of benchmark datasets. Experiments show that our algorithm can give very accurate solutions. It outperforms most of the existing programs when the error rate of the input fragments is high.

G-SNPM - A GPU-based SNP mapping tool

G-SNPM - A GPU-based SNP mapping tool

A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics

A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc‐finger nucleases

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C.intestinalis genome because this locus is neutral for the development and growth of C.intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C.intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C.intestinalis.

DNA‐Based Identification of Forensically ImportantLucilia(Diptera: Calliphoridae) in the Continental United States*

Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA-based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100-bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA-based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA-based identification is likely to be successful for the other seven species.

Primers and Probes Development for Specific PCR Detection of Genetically Modified Common Bean (Phaseolus vulgaris) Embrapa 5.1

The genetically modified common bean Embrapa 5.1, developed by Brazilian Agricultural Research Corporation (Embrapa), is the first commercial GM plant produced in Latin America. It presents high resistance to the Bean golden mosaic virus. In this work, primers and probes targeting a taxon-specific reference DNA sequence for the common bean (Phaseolus vulgaris L.) and a construct-specific DNA sequence of Embrapa 5.1 GM common bean were successfully developed. The primers and probes showed high specificity for the target detection. Both methods showed suitable efficiency and performance to be used as an endogenous target for detection of common bean DNA and for construct-specific detection of GM common bean Embrapa 5.1, respectively. Both real-time PCR assays proved to be valuable for future assessment of interlaboratory studies.

BarraCUDA - a fast short read sequence aligner using graphics processing units

BackgroundWith the maturation of next-generation DNA sequencing (NGS) technologies, the throughput of DNA sequencing reads has soared to over 600 gigabases from a single instrument run. General purpose computing on graphics processing units (GPGPU), extracts the computing power from hundreds of parallel stream processors within graphics processing cores and provides a cost-effective and energy efficient alternative to traditional high-performance computing (HPC) clusters. In this article, we describe the implementation of BarraCUDA, a GPGPU sequence alignment software that is based on BWA, to accelerate the alignment of sequencing reads generated by these instruments to a reference DNA sequence.FindingsUsing the NVIDIA Compute Unified Device Architecture (CUDA) software development environment, we ported the most computational-intensive alignment component of BWA to GPU to take advantage of the massive parallelism. As a result, BarraCUDA offers a magnitude of performance boost in alignment throughput when compared to a CPU core while delivering the same level of alignment fidelity. The software is also capable of supporting multiple CUDA devices in parallel to further accelerate the alignment throughput.ConclusionsBarraCUDA is designed to take advantage of the parallelism of GPU to accelerate the alignment of millions of sequencing reads generated by NGS instruments. By doing this, we could, at least in part streamline the current bioinformatics pipeline such that the wider scientific community could benefit from the sequencing technology.BarraCUDA is currently available from http://seqbarracuda.sf.net

Egg forensics: An appraisal of DNA sequencing to assist in species identification of illegally smuggled eggs

Psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. The illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically ‘pollute’ local native avifauna. To reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. This creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. Here, we use 99 avian eggs seized from carriers coming into and within Australia, as a result of suspected illegal trade. We investigate and evaluate the use of mitochondrial DNA (mtDNA) to accurately identify eggs to family, genus or species level. However, Identification of a species based on percentage mtDNA similarities is difficult without good representations of the inter- and intra-levels of species variation. Based on the available reference database, we were able to identify 52% of the eggs to species level. Of those, 10 species from eight genera were detected, all of which belong to the parrot (Psittacidae) and cockatoo (Cacatuidae) families. Of the remaining 48%, a further 36% of eggs were identified to genus level, and 12% identified to family level using our assignment criteria. Clearly the lack of validated DNA reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtDNA reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny.

Sequencing of high G+C microbial genomes using the ultrafast pyrosequencing technology

Next generation pyrosequencing of high G+C content genomes still poses problems to automated sequencing and assembly processes which necessitates cost and time intensive manual work in order to finish such genomes completely. The sequencing of the high G+C actinomycete Actinoplanes sp. SE50/110 was performed with standard pyrosequencing technology (454 Life Sciences) and revealed a high number of gaps. The reasons for the introduction of gaps were analyzed on a previously known 41kb long DNA reference sequence from Actinoplanes sp. SE50/110, hosting the acarbose biosynthesis gene cluster. Mapping of the sequencing results on the reference gene cluster sequence revealed a fragmentation into 30 contiguous sequences of different lengths. The gaps between these sequences were characterized by extremely low read coverage which strongly correlated with the G+C content in the gap regions in a negative manner. Furthermore, the gap-sequences contained strong stem-loop structures which hindered the amplification of these sequences during the emulsion PCR. Being significantly underrepresented or absent in the subsequent sequencing process, these sequences lead to weakly or uncovered genomic regions which forces the assembly algorithm to output multiple contiguous sequences instead of one finished genome. However, by applying a different pyrosequencing protocol, it was possible to sequence the complete acarbose biosynthesis gene cluster. The changes to the protocol include longer read length and addition of chemicals to the amplification chemistry, which reduces the self-annealing of DNA fragments during the amplification process and enables the complete reconstruction of high G+C content genomes without manual intervention.

Letter to the Editor Boning up on mutations: assessing the significance of candidate disease-causing DNA sequence variation

I write to you regarding the paper by Yang et al. (2011) entitled “Mutation characteristics in type I collagen genes in Chinese patients with osteogenesis imperfecta”, which was recently published in Genetics and Molecular Research. The impression given by the authors is that they have identified disease-causing DNA sequence variants in COL1A1 and COL1A2, which result in osteogenesis imperfecta (OI) in five of the probands studied. However, the data presented in the paper do not support such a proposition and I will discuss each variant in turn. However, it is important to first clarify the identity of the relevant reference DNA sequences and variant reporting conventions.