scholarly journals HiMAP: robust Phylogenomics from Highly Multiplexed Amplicon sequencing

2017 ◽  
Author(s):  
Julian R. Dupuis ◽  
Forest T. Bremer ◽  
Angela Kauwe ◽  
Michael San Jose ◽  
Luc Leblanc ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Insect Pests ◽  
Amplicon Sequencing ◽  
Read Mapping ◽  
Sister Relationship ◽  
New Approach ◽  
Hands On ◽  
Locus Selection ◽  
Molecular Phylogenetic ◽  
Tephritid Fruit Flies

ABSTRACTHigh-throughput sequencing has fundamentally changed how molecular phylogenetic datasets are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and post-sequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically-important agricultural insect pests. The resulting dataset (>150,000 bp concatenated alignment) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status ofZeugodacusand the sister relationship betweenDacusandZeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting dataset provides into relationships of this diverse insect group.

scholarly journals Phylogenomics and barcoding ofPanax: toward the identification of ginseng species

2018 ◽  
Author(s):  
V. Manzanilla ◽  
A. Kool ◽  
Nhat L. Nguyen ◽  
H. Nong Van ◽  
H. Le Thi Thu ◽  
...  
Keyword(s):  
Panax Ginseng ◽  
Molecular Identification ◽  
High Throughput Sequencing ◽  
Economic Value ◽  
Cost Effective ◽  
Morphological Characters ◽  
Species Discrimination ◽  
Sister Relationship ◽  
Plastid Genomes ◽  
Molecular Phylogenetic

AbstractBackgroundThe economic value of ginseng in the global medicinal plant trade is estimated to be in excess of US$2.1 billion. At the same time, the evolutionary placement of ginseng (Panax ginseng) and the complex evolutionary history of the genus is poorly understood despite several molecular phylogenetic studies. In this study, we use a full plastome phylogenomic framework to resolve relationships inPanaxand to identify molecular markers for species discrimination.ResultsWe used high-throughput sequencing of MBD2-Fc fractionatedPanaxDNA to supplement publicly available plastid genomes to create a phylogeny based on fully assembled and annotated plastid genomes from 60 accessions of 8 species. The plastome phylogeny based on a 163 kbp matrix resolves the sister relationship ofPanax ginsengwithP. quinquefolius. The closely related speciesP. vietnamensisis supported as sister ofP. japonicus. The plastome matrix also shows that the markerstrnC-rps16, trnS-trnG, andtrnE-trnMcould be used for unambiguous molecular identification of all the represented species in the genus.ConclusionsMBD2 depletion reduces the cost of plastome sequencing, which makes it a cost-effective alternative to Sanger sequencing based DNA barcoding for molecular identification. The plastome phylogeny provides a robust framework that can be used to study the evolution of morphological characters and biosynthesis pathways of ginsengosides for phylogenetic bioprospecting. Molecular identification of ginseng species is essential for authenticating ginseng in international trade and it provides an incentive for manufacturers to create authentic products with verified ingredients.

scholarly journals Analysis of the Fungal Community in Ziziphi Spinosae Semen through High-Throughput Sequencing

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 494 ◽  
Author(s):  
Mengyue Guo ◽  
Wenjun Jiang ◽  
Jiaoyang Luo ◽  
Meihua Yang ◽  
Xiaohui Pang
Keyword(s):  
Fungal Community ◽  
High Throughput Sequencing ◽  
Drug Efficacy ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Fungal Communities ◽  
Consumer Health ◽  
Efficacy And Safety ◽  
Fungal Contamination ◽  
New Approach

Ziziphi Spinosae Semen (ZSS) has been widely used in traditional Chinese medicine system for decades. Under proper humidity and temperature, ZSS is easily contaminated by fungi and mycotoxins during harvest, storage, and transport, thereby posing a considerable threat to consumer health. In this study, we first used the Illumina MiSeq PE250 platform and targeted the internal transcribed spacer 2 sequences to investigate the presence of fungi in moldy and normal ZSS samples collected from five producing areas in China. Results showed that all 14 samples tested were contaminated by fungi. Ascomycota was the dominant fungus at the phylum level, accounting for 64.36–99.74% of the fungal reads. At the genus level, Aspergillus, Candida, and Wallemia were the most predominant genera, with the relative abundances of 13.52–87.87%, 0.42–64.56%, and 0.06–34.31%, respectively. Meanwhile, 70 fungal taxa were identified at the species level. Among these taxa, three potential mycotoxin-producing fungi, namely, Aspergillus flavus, A. fumigatus, and Penicillium citrinum that account for 0.30–36.29%, 0.04–7.37%, and 0.01–0.80% of the fungal reads, respectively, were detected in all ZSS samples. Moreover, significant differences in fungal communities were observed in the moldy and normal ZSS samples. In conclusion, our results indicated that amplicon sequencing is feasible for the detection and analysis of the fungal community in the ZSS samples. This study used a new approach to survey the fungal contamination in herbal materials. This new approach can provide early warning for mycotoxin contamination in herbal materials, thereby ensuring drug efficacy and safety.

scholarly journals Extremely Halophilic Biohydrogen Producing Microbial Communities from High-Salinity Soil and Salt Evaporation Pond

Fuels ◽  
2021 ◽  
Vol 2 (2) ◽  
pp. 241-252
Author(s):  
Dyah Asri Handayani Taroepratjeka ◽  
Tsuyoshi Imai ◽  
Prapaipid Chairattanamanokorn ◽  
Alissara Reungsang
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
High Salinity ◽  
Amplicon Sequencing ◽  
Spatial Proximity ◽  
Lignocellulosic Waste ◽  
Evaporation Pond ◽  
Operational Taxonomic Units ◽  
Determining Factor

Extreme halophiles offer the advantage to save on the costs of sterilization and water for biohydrogen production from lignocellulosic waste after the pretreatment process with their ability to withstand extreme salt concentrations. This study identifies the dominant hydrogen-producing genera and species among the acclimatized, extremely halotolerant microbial communities taken from two salt-damaged soil locations in Khon Kaen and one location from the salt evaporation pond in Samut Sakhon, Thailand. The microbial communities’ V3–V4 regions of 16srRNA were analyzed using high-throughput amplicon sequencing. A total of 345 operational taxonomic units were obtained and the high-throughput sequencing confirmed that Firmicutes was the dominant phyla of the three communities. Halanaerobium fermentans and Halanaerobacter lacunarum were the dominant hydrogen-producing species of the communities. Spatial proximity was not found to be a determining factor for similarities between these extremely halophilic microbial communities. Through the study of the microbial communities, strategies can be developed to increase biohydrogen molar yield.

scholarly journals Noteworthy record of the Ethiopian genet, Genetta abyssinica, (Carnivora, Viverridae) from Djibouti informs its phylogenetic position within Genetta

Mammalia ◽  
2019 ◽  
Vol 83 (2) ◽  
pp. 180-189 ◽  
Author(s):  
Adam W. Ferguson ◽  
Houssein R. Roble ◽  
Molly M. McDonough
Keyword(s):  
Phylogenetic Relationships ◽  
Sequence Data ◽  
Alternative Hypothesis ◽  
Phylogenetic Analyses ◽  
Phylogenetic Position ◽  
Sister Relationship ◽  
Molecular Sequence Data ◽  
Molecular Sequence ◽  
Molecular Phylogenetic ◽  
Relationship Of

AbstractThe molecular phylogeny of extant genets (Carnivora, Viverridae,Genetta) was generated using all species with the exception of the Ethiopian genetGenetta abyssinica. Herein, we provide the first molecular phylogenetic assessment ofG. abyssinicausing molecular sequence data from multiple mitochondrial genes generated from a recent record of this species from the Forêt du Day (the Day Forest) in Djibouti. This record represents the first verified museum specimen ofG. abyssinicacollected in over 60 years and the first specimen with a specific locality for the country of Djibouti. Multiple phylogenetic analyses revealed conflicting results as to the exact relationship ofG. abyssinicato otherGenettaspecies, providing statistical support for a sister relationship to all other extant genets for only a subset of mitochondrial analyses. Despite the inclusion of this species for the first time, phylogenetic relationships amongGenettaspecies remain unclear, with limited nodal support for many species. In addition to providing an alternative hypothesis of the phylogenetic relationships among extant genets, this recent record provides the first complete skeleton of this species to our knowledge and helps to shed light on the distribution and habitat use of this understudied African small carnivore.

scholarly journals Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Francesca De Filippis ◽  
Manolo Laiola ◽  
Giuseppe Blaiotta ◽  
Danilo Ercolini
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Biological Samples ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Fungal Species ◽  
Fungal Communities ◽  
Its2 Region ◽  
Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

scholarly journals Combining whole genome shotgun sequencing and rDNA amplicon analyses to improve detection of microbe-microbe interaction networks in plant leaves

2019 ◽  
Author(s):  
Julian Regalado ◽  
Derek S. Lundberg ◽  
Oliver Deusch ◽  
Sonja Kersten ◽  
Talia Karasov ◽  
...  
Keyword(s):  
16S Rdna ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Low Complexity ◽  
Shotgun Sequencing ◽  
Wild Plants ◽  
Hybrid Strategy ◽  
Domains Of Life ◽  
Metagenome Sequencing

AbstractMicroorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, acquire nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of 16S rDNA and/or the internal transcribed spacer (ITS) of rDNA loci, but the decreasing cost of high-throughput sequencing has made shotgun metagenome sequencing increasingly accessible. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S rDNA and eukaryotic ITS1 amplicon data from 176 of these samples. The shotgun data were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. For shotgun and amplicon data, microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. We use the metagenome data, which captures the ratio of bacterial to plant DNA in leaves of wild plants, to scale the 16S rDNA amplicon data such that they reflect absolute bacterial abundance. We show that this cost-effective hybrid strategy overcomes compositionality problems in amplicon data and leads to fundamentally different conclusions about microbiome community assembly.

scholarly journals Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Marius Welzel ◽  
Anja Lange ◽  
Dominik Heider ◽  
Michael Schwarz ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Workflow Management ◽  
Amplicon Sequencing ◽  
Version Control ◽  
Marker Genes ◽  
Sequencing Data ◽  
Taxonomic Assignment ◽  
Ecological Processes ◽  
Link Type ◽  
User Friendly

Abstract Background Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. Results We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix) or as a Docker container on DockerHub (https://hub.docker.com/r/mw55/natrix). Conclusion Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Targeting detoxification genes by phloem-mediated RNAi: A new approach for controlling phloem-feeding insect pests

2018 ◽  
Vol 100 ◽  
pp. 10-21 ◽  
Author(s):  
Galit Eakteiman ◽  
Rita Moses-Koch ◽  
Pnina Moshitzky ◽  
Natividad Mestre-Rincon ◽  
Daniel G. Vassão ◽  
...  
Keyword(s):  
Insect Pests ◽  
New Approach ◽  
Phloem Feeding

scholarly journals Synthetic Sequencing Standards: A Guide to Database Choice for Rumen Microbiota Amplicon Sequencing Analysis

2020 ◽  
Vol 11 ◽  
Author(s):  
Paul E. Smith ◽  
Sinead M. Waters ◽  
Ruth Gómez Expósito ◽  
Hauke Smidt ◽  
Ciara A. Carberry ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Gas Production ◽  
Reference Database ◽  
Specific Reference ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Rumen Microbiota ◽  
Reference Databases

Our understanding of complex microbial communities, such as those residing in the rumen, has drastically advanced through the use of high throughput sequencing (HTS) technologies. Indeed, with the use of barcoded amplicon sequencing, it is now cost effective and computationally feasible to identify individual rumen microbial genera associated with ruminant livestock nutrition, genetics, performance and greenhouse gas production. However, across all disciplines of microbial ecology, there is currently little reporting of the use of internal controls for validating HTS results. Furthermore, there is little consensus of the most appropriate reference database for analyzing rumen microbiota amplicon sequencing data. Therefore, in this study, a synthetic rumen-specific sequencing standard was used to assess the effects of database choice on results obtained from rumen microbial amplicon sequencing. Four DADA2 reference training sets (RDP, SILVA, GTDB, and RefSeq + RDP) were compared to assess their ability to correctly classify sequences included in the rumen-specific sequencing standard. In addition, two thresholds of phylogenetic bootstrapping, 50 and 80, were applied to investigate the effect of increasing stringency. Sequence classification differences were apparent amongst the databases. For example the classification of Clostridium differed between all databases, thus highlighting the need for a consistent approach to nomenclature amongst different reference databases. It is hoped the effect of database on taxonomic classification observed in this study, will encourage research groups across various microbial disciplines to develop and routinely use their own microbiome-specific reference standard to validate analysis pipelines and database choice.

A new family of Laniatores (Arachnida:Opiliones) from the Afrotropics

2011 ◽  
Vol 25 (2) ◽  
pp. 143 ◽  
Author(s):  
Prashant P. Sharma ◽  
Carlos E. Prieto ◽  
Gonzalo Giribet
Keyword(s):  
Canary Islands ◽  
Morphological Evidence ◽  
Monotypic Genus ◽  
Sister Relationship ◽  
New Family ◽  
Phylogenetic Studies ◽  
Biogeographic Provinces ◽  
The Family ◽  
Molecular Phylogenetic

Among Opiliones, Afrotropical lineages constitute some of the least studied groups in comparison with those endemic to other biogeographic provinces. Based upon morphological evidence, we erect Pyramidopidae, fam. nov. to distinguish a group of Laniatores from the family Phalangodidae. We review evidence from recent molecular phylogenetic studies that corroborate the independence of Pyramidopidae, fam. nov. from previously described families and support its sister relationship to another largely Afrotropical group, the family Assamiidae. The monotypic genus Maiorerus Rambla, 1993 is transferred to Pyramidopidae, fam. nov. The new family comprises 12 genera geographically restricted to Africa and the adjacent Canary Islands. Interfamilial relationships of the derived Laniatores are discussed in the context of gross and genitalic morphology.

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