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2.
J Biomol Tech ; 30(Suppl): S43, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31896970

RESUMO

The ABRF Next-Generation Sequencing (NGS) Study examines well-characterized human and bacterial genomic reference material to evaluate the reproducibility of sequence data generated using industry standard DNA sequencing platforms. This study is not a "bake-off" between instruments, but rather an effort to establish a reference dataset that will allow users of high-throughput platforms to evaluate the performance of their instruments as protocols and technologies rapidly evolve. In collaboration with the Genome in a Bottle Consortium and other sequencing community stakeholders, the ABRF-NGS group has sequenced NIST DNA Reference Materials derived from cell lines of Personal Genome Project (PGP) contributors, as well as ATCC bacterial genomic microbiome standards, using a range of technologies including Illumina and Thermo Fischer. For each instrument, the study design included inter- and intra-lab replicates to facilitate different levels of output comparison. All data were processed through an alignment and variant analysis pipeline using Sentieon, and examined for sequencing accuracy, quality divergences, and performance within genomic contexts such as GC extremes, areas of low complexity, and "difficult" regions of the genome that include the "Classes of Evil". Different platforms exhibited relative strengths and weaknesses relative to one another and with respect to different samples. These comparisons, which are available in a dynamic, publicly accessible web resource, can be used as a baseline for researchers to evaluate their own NGS outputs.

3.
Genome Biol ; 18(1): 182, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934964

RESUMO

BACKGROUND: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. RESULTS: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. CONCLUSIONS: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.


Assuntos
Benchmarking/métodos , Mapeamento de Sequências Contíguas/métodos , Código de Barras de DNA Taxonômico/métodos , Metagenoma , Análise de Sequência de DNA/métodos , Software , Benchmarking/normas , Mapeamento de Sequências Contíguas/normas , Código de Barras de DNA Taxonômico/normas , Humanos , Microbiota , Filogenia , Análise de Sequência de DNA/normas
4.
Reprod Toxicol ; 69: 174-186, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28267574

RESUMO

Evolutionary thinking continues to challenge our views on health and disease. Yet, there is a communication gap between evolutionary biologists and toxicologists in recognizing the connections among developmental pathways, high-throughput screening, and birth defects in humans. To increase our capability in identifying potential developmental toxicants in humans, we propose to apply evolutionary genetics to improve the experimental design and data interpretation with various in vitro and whole-organism models. We review five molecular systems of stress response and update 18 consensual cell-cell signaling pathways that are the hallmark for early development, organogenesis, and differentiation; and revisit the principles of teratology in light of recent advances in high-throughput screening, big data techniques, and systems toxicology. Multiscale systems modeling plays an integral role in the evolutionary approach to cross-species extrapolation. Phylogenetic analysis and comparative bioinformatics are both valuable tools in identifying and validating the molecular initiating events that account for adverse developmental outcomes in humans. The discordance of susceptibility between test species and humans (ontogeny) reflects their differences in evolutionary history (phylogeny). This synthesis not only can lead to novel applications in developmental toxicity and risk assessment, but also can pave the way for applying an evo-devo perspective to the study of developmental origins of health and disease.


Assuntos
Evolução Molecular , Medição de Risco , Teratologia , Animais , Biologia Computacional , Humanos , Filogenia , Biologia de Sistemas
5.
J Biomol Tech ; 28(1): 31-39, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337070

RESUMO

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.


Assuntos
Microbiologia Ambiental , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ambientes Extremos , Metagenoma , Tipagem Molecular/normas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA/normas
6.
Mitochondrial DNA B Resour ; 1(1): 425-427, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27617305

RESUMO

We report the extraction of a bed bug mitogenome from high-throughput sequencing projects originally focused on the nuclear genome of Cimex lectularius. The assembled mitogenome has a similar AT nucleotide composition bias found in other insects. Phylogenetic analysis of all protein-coding genes indicates that C. lectularius is clearly a member of a paraphyletic Cimicomorpha clade within the Order Hemiptera.

7.
Nat Commun ; 7: 10164, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26836631

RESUMO

The common bed bug (Cimex lectularius) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bug's basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations.


Assuntos
Percevejos-de-Cama/genética , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida/genética , Animais , Sangue , Mapeamento Cromossômico , Ingestão de Alimentos , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Modelos Moleculares , Filogenia , Análise de Sequência de RNA
8.
Data Brief ; 6: 279-81, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26862572

RESUMO

Twenty one fully sequenced and well annotated insect genomes were used to construct genome content matrices for phylogenetic analysis and functional annotation of insect genomes. To examine the role of e-value cutoff in ortholog determination we used scaled e-value cutoffs and a single linkage clustering approach.. The present communication includes (1) a list of the genomes used to construct the genome content phylogenetic matrices, (2) a nexus file with the data matrices used in phylogenetic analysis, (3) a nexus file with the Newick trees generated by phylogenetic analysis, (4) an excel file listing the Core (CORE) genes and Unique (UNI) genes found in five insect groups, and (5) a figure showing a plot of consistency index (CI) versus percent of unannotated genes that are apomorphies in the data set for gene losses and gains and bar plots of gains and losses for four consistency index (CI) cutoffs.

9.
Mitochondrial DNA A DNA Mapp Seq Anal ; 27(4): 2826-32, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26104159

RESUMO

Six complete and three partial actiniarian mitochondrial genomes were amplified in two semi-circles using long-range PCR and pyrosequenced in a single run on a 454 GS Junior, doubling the number of complete mitogenomes available within the order. Typical metazoan mtDNA features included circularity, 13 protein-coding genes, 2 ribosomal RNA genes, and length ranging from 17,498 to 19,727 bp. Several typical anthozoan mitochondrial genome features were also observed including the presence of only two transfer RNA genes, elevated A + T richness ranging from 54.9 to 62.4%, large intergenic regions, and group 1 introns interrupting NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit I, the latter of which possesses a homing endonuclease gene. Within the sea anemone Alicia sansibarensis, we report the first mitochondrial gene order rearrangement within the Actiniaria, as well as putative novel non-canonical protein-coding genes. Phylogenetic analyses of all 13 protein-coding and 2 ribosomal genes largely corroborated current hypotheses of sea anemone interrelatedness, with a few lower-level differences.


Assuntos
DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Anêmonas-do-Mar/genética , Animais , Filogenia , RNA de Transferência/genética , Anêmonas-do-Mar/classificação
10.
Mol Phylogenet Evol ; 97: 224-232, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26549428

RESUMO

Twenty-one fully sequenced and well annotated insect genomes were examined for genome content in a phylogenetic context. Gene presence/absence matrices and phylogenetic trees were constructed using several phylogenetic criteria. The role of e-value on phylogenetic analysis and genome content characterization is examined using scaled e-value cutoffs and a single linkage clustering approach to orthology determination. Previous studies have focused on the role of gene loss in terminals in the insect tree of life. The present study examines several common ancestral nodes in the insect tree. We suggest that the common ancestors of major insect groups like Diptera, Hymenoptera, Hemiptera and Holometabola experience more gene gain than gene loss. This suggests that as major insect groups arose, their genomic repertoire expanded through gene duplication (segmental duplications), followed by contraction by gene loss in specific terminal lineages. In addition, we examine the functional significance of the loss and gain of genes in the divergence of some of the major insect groups.


Assuntos
Evolução Molecular , Genoma de Inseto/genética , Insetos/classificação , Insetos/genética , Anotação de Sequência Molecular , Filogenia , Animais , Duplicação Gênica/genética , Genômica
11.
BMC Genomics ; 16: 840, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26494377

RESUMO

BACKGROUND: The Myxozoa, a group of oligocellular, obligate endoparasites, has long been poorly understood in an evolutionary context. Recent genome-level sequencing techniques such as RNA-seq have generated large amounts of myxozoan sequence data, providing valuable insight into their evolutionary history. However, sequences from host tissue contamination are present in next-generation sequencing reactions of myxozoan tissue, and differentiating between the two has been inadequately addressed. In order to shed light on the genetic underpinnings of myxozoan biology, assembled contigs generated from these studies that derived from the myxozoan must be decoupled from transcripts derived from host tissue and other contamination. This study describes a pipeline for categorization of transcripts asmyxozoan based on similarity searching with known host and parasite sequences, explores the extent to which host contamination is present in previously existing myxozoan datasets, and implements this pipeline on a newly sequenced transcriptome of Myxobolus pendula, a parasite of the common creek chub gill arch. METHODS: The insilico hybridization pipeline uses iterative BLAST searching and database-driven e-value comparison to categorize transcripts as deriving from host, parasite, or other contamination. Functional genetic analysis of M. pendula was conducted using further BLAST searching, Hidden Markov Modeling, and sequence alignment and phylogenetic reconstruction. RESULTS: Three RNA libraries of encysted M. pendula plasmodia were sequenced and subjected to the method. Nearly half of the final set of contiguous assembly sequences (47.3 %) was identified as putative myxozoan transcripts. Putative contamination was also identified in at least 1/3(rd) of previously published myxozoan transcripts. The set of M. pendula transcripts was mined for a range of biologically insightful genes, including taxonomically restricted nematocyst structural proteins and nematocyst proteins identified through mass tandem spectrometry of other cnidarians. Several novel findings emerged, including a fourth myxozoan minicollagen gene, putative myxozoan toxin proteins,and extracellular matrix glycoproteins. CONCLUSIONS: This study serves as a model for the handling of next-generation myxozoan sequence. The need for careful categorization was demonstrated in both previous and new sets of myxozoan sequences. The final set of confidently assigned myxozoan transcripts can be mined for any biologically relevant gene or gene family without spurious misidentification of host contamination as a myxozoan homolog. As exemplified by M. pendula, the repertoire of myxozoan polar capsules may be more complex than previously thought, with an additional minicollagen homolog and putative expression of toxin proteins.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Myxozoa/genética , Filogenia , Transcriptoma/genética , Animais , Simulação por Computador , Genoma , Toxinas Biológicas/genética
12.
J Parasitol ; 101(3): 269-74, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25621522

RESUMO

Myxozoans are a clade of highly derived cnidarians. The phylogenetic identity of these extremely simplified parasites of aquatic vertebrates and invertebrates had long been uncertain, with all early classifications designating Myxozoa as protists. Though suggestions were frequently made that the infective spores of these parasites are multicellular and possibly of cnidarian origin, it would take a phylogenetic analysis of ultrastructural developmental characters in combination with rRNA gene sequences to verify the Myxozoa as secondarily reduced cnidarians, sister to the polypoidozoan parasite Polypodium hydriforme . While a series of subsequent molecular studies suggested hypotheses of Myxozoa as basal bilaterians, triploblasts, or even nematodes, phylogenomic analyses with improved taxon sampling corroborated the landmark paper that verified the cnidarian nature of this group. This review of the body of phylogenetic work on Myxozoa aims to clarify historical progress and current knowledge, as well as to emphasize the opportune position that myxozoan biologists now are in, to address fundamental questions of cell biology of these parasites as well as the evolution of animal life.


Assuntos
Cnidários/classificação , Doenças Parasitárias/história , Doenças Parasitárias/parasitologia , Filogenia , Animais , Cnidários/genética , História do Século XIX , História do Século XX , História do Século XXI , Myxozoa/classificação , Myxozoa/genética
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