Progress 10/01/13 to 09/30/17
Outputs
Target Audience:The target audience for this project includes a variety of researchers from diverse fields including nematology, parasitology, molecular biology, microbiology, evolutionary biology, agriculturem pest management, and bioinformatics and computer science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Opportunities for professional development from this grant have included the training of two graduate students in molecular biology, next-generation sequencing, and bioinformatics analysis. Furthermore, the data generated by this project is used in ongoing bioinformatics workshops (various sponsors, including NSF and NIH) and in bioinformatics for curriculum development across the state of New Hampshire as part of our NH-INBRE grant (NIH). These training programs and workshops have wide-reaching impacts for UNH, as well as the primarily undergraduate institutions in the state of NH and for a HBCU partner in North Carolina. Because the tools and pipelines for bioinformatics analyses are continually changing, a well-characterized data set such as these Anguina and the associated microbiome data are an ideal training set for assembly and annotation pipelines. These data also provide good comparator metrics for refining bioinformatics analyses and experimental design. How have the results been disseminated to communities of interest?The workshop training and curriculum integration described above are one method of dissemination. Our newly added collaborator is an expert in this particular nematode group and is providing some additional guidance in refining the current dataset for publication. The genome manuscript is close to submission, and we are working with NCBI to get the read data and the genome archived appropriately so it is accessible to the broader research community. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This research gocuses on the interactions between a plant parasititc nematode and its associated bacterial community. To accomplish these aims, we have foucsed on the genome of Anguina tritici, a plant parasitic nematode and its associated microbiome. The Anguina genus is a member of the same subfamily of nematodes (Anguininae) as Ditylenchus (as originally proposed). Using next generation sequencing and molecular genomics techniques, we have developed a draft genome for Anguina tritici, and conducted a microbiome survey of the dessicated juvenile A. tritici as isolated from the infected wheat galls. From 8 galls, we sequenced the associated nematodes, producing 8 draft genomes for Anguina tritici. The sequence was assembled into a draft genome for Anguina tritici. The mitochondrial genome for these nematodes was interesting in that it seems to be tripartite rather than a single molecule. Experiments to confirm the presence of three circular molecules for the mitochondrial genome are in progress. Also, several associated bacterial genomes were also present. The associated microbial community was assayed using the V4 region of the 16S small subunit ribosomal RNA gene. The presence of the bacterium Wolbachia in two of the eight samples was of particular interest and should be studied further, and may provide insight into the mechanisms of survival and parasitismin this group of nematodes.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:This work is of interest to the agricultural community and those interested in pest-management. Other target audience include students and researchers interest in understanding the genomics and microbiomes of plant parasites. Data generated by this project was incorporated into several NSF sponsored workshops focused on bioinformatics training as well as in bioinformatics curriculum development for NH_INBRE project sponsored by NIH. Changes/Problems:The difficulties obtaining appropriate Ditylenchus dipsaci nematode samples continue, and we are working with a potential source/collaborator to obtain DNA of sufficient quality and quantity for genomic sequencing. However, the data in progress is from a nematode in the same order as Ditylenchus, and should be informative for the Ditylenchus assembly and analyses. What opportunities for training and professional development has the project provided?The data generated by this project is used in ongoing bioinformatics workshops (various sponsors, including NSF) and bioinformatics for curriculum development for our NH-INBRE grant (NIH supported). These training programs and workshops have wide-reaching impacts for UNH, as well as the primarily undergraduate institutions in the state of NH and for a HBCU partner in North Carolina. Because the tools and pipelines for bioinformatics analyses are continually evolving, a well-characterized dataset such as these Anguina data, are an ideal training set for assembly and annotation pipelines, and provide good comparator metrics for refining analyses and experimental design. How have the results been disseminated to communities of interest?The workshops and trainings described above are one method of dissemination. We have recently contacted a potential collaborator and expert on this particular nematode genus, and he has provided some additional data to refine the current analysis and assembly and will help us design some future experiments to better understand the molecular biology of these seed gall nematodes. The genome manuscript is in preparation, and the data will be submitted to the NBCI archives and other appropriate sites (i.e. wormbase), as we move forward and in conjunction with the manuscript submission/publication. What do you plan to do during the next reporting period to accomplish the goals?We have been in contact with several Ditylenchus (garlic bloat) nematode researchers and are trying to determine best methods to obtain DNA from Ditylenchus without having them ship the actual parasitic nematodes. We will continue our work on the Anguina life cycle analyses by generating targeted RNA-seq datasets, and working to better characterize and understand our Wolbachia analyses.
Impacts
What was accomplished under these goals?
This research focuses on the interactions between a plant parasitic nematode and its associated bacterial community. We focus on the microbiome of the seed gall nematode, an invasive pest of wheat and related crops. The second focus is on the microbial community of the soil associated with the plant parasitic nematode and its host. We hypothesize that a subset of the associated bacterial community for the nematode is critical to its life history. We will compare the nematode-associated bacterial communities with the local "free-living" communities and assess how those vary within and between nematode populations. This information will be critical to informed management of these environments and the development of effective pest control practices. During the past year, we have been refining our analyses of the Anguina tritici genome and microbiome sequences that we generated during the previous year. The manuscript is in preparation, and the data is being prepared for submission to the NCBI archives. We have contacted a colleague (Dr. Sergei Subbotin--California Dept. of Food and Agriculture) who has some unpublished RNA-seq data for Anguina tritici and was willing to share that data, so it is currently being incorporated to better refine the gene models predicted by the genome assembly. The estimated genome size for Anguina tritici is approximately 185M bases, with a tripartite mitochondrial genome. Also, the mitochondrial genome appears to code for ATPase 8, which is absent in many nematode species. Benchmarking Universal Single-Copy Orthologs (BUSCO) analyses indicate the present of ~210 complete, single copy orthologs from 429 searched, and approximately 140 or so that are not found. Associated bacterial strains identified include Wolbachia, Marinomonas, and several representatives of Bacillus. Furthermore, we are working to develop a set of RNA-seq experiments for Anguina tritici to better understand the unique traits of this seed gall nematode species and its microbiome. The analysis of the microbiome data has also provided some interesting insights, including a potentially unique strain of the Wolbachia pathogen, which we are investigating further. Wolbachia has an interesting, demonstrated biocontrol capability, and if we have indeed identified a new strain, further analyses and research plans will need to be developed.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience for this project includes a variety of researchers from diverse fields including nematology, parasitology, molecular biology, microbiology, evolutionary biology, agriculture, pest management, and bioinformatics/computer science. Workshops which integrated some of the data generated as part of this proposal included an NSF-funded Bioinformatics Bootcamp (Summer 2015), which included 20 undergraduate participants from various colleges and universities across the U.S. Other ongoing workshops include those supported by an NIH-INBRE grant which make use of the data and methods developed as part of this grant include data analysis workshops integrating bioinformatics for data generated by the specific molecular methods used in this proposal including experimental design and high-throughput sequencing. During the summer, the Thomas lab also hosted two high school students who worked with some of the data sets. Courses which included the data and methodology as part of their curriculum included a primarily senior level/graduate student level course called Applied Bioinformatics and a Programming for Biologists course. The Applied Bioinformatics course brings together students from the biological sciences and the computer science programs to work together on data analysis and pipeline development challenges. The Programming for Biologists course teaches command line skills as well as Python coding skills for use in bioinformatics projects. Changes/Problems:We initially had difficulty in obtaining the Ditylenchus dipsaci specimens, but that was addressed during the summer 2015 Nematology meetings, and we hope to have all the specimens in hand to add to the datasets we are currently generating for other plant parasitic nematodes. The development of a modified protocol for generating libraries for high-throughput sequencing and metagenomics is also major, unanticipated result that will significantly reduce the costs associated with this project and should enable us to expand our analyses beyond the proposed species. What opportunities for training and professional development has the project provided?Workshops supported this year included the 2015 Summer Bioinformatics bootcamp and various bioinformatics and molecular biology workshops at our partner INBRE schools. Courses which used data from this project include Applied Bioinformatics and Programming for Biologists. During the summer, two high school students conducted research in the lab and worked with the data from this project and learned some of the molecular techniques used in generating the DNA sequences to implement in their own projects. Two graduate students (Joe Sevigny, Genetics and Jordan Ramsdell, Computer Science) worked with PI Thomas and the Research Supervisor (Krystalynne Morris) on the analysis of the data. A technician (Stephen Simpson) was also involved in the molecular work on these specimens. How have the results been disseminated to communities of interest?Outreach activities have included the workshops and courses mentioned in the previous sections. We are actively engaging with our professional colleagues (nematologists, microbiologists and bioinformaticists) as we are analyzing this current dataset and working to generate the Ditylenchus data sets. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we anticipate disseminating the data generated for the Anguina tritici specimens at the annual meeting of the Society of Nematologists, as well as generating a manuscript to with our description of the shotgun sequence data, assembly, and analysis of the microbial associates. The data will be deposited in the appropriate public repositories, including GenBank and we will work with other groups such as WormBase and Nematode.net to make the data accessible to interested researchers.
Impacts
What was accomplished under these goals?
One of the proposed species for this grant is the parasitic nematode Ditylenchus dipsaci, and we were able to engage researchers in that community during the annual Society of Nematologists meeting in July 2015. We are currently receiving and preparing specimens for template generation and sequencing analyses. Objective: Generate whole genome population genetic analyses in important plant parasitic nematodes. During the past year, we were fortunate to obtain specimens of a related group of nematodes, Anguina tritici, which are in the same order as Ditylenchus (Tylenchida). The specimens came to us in the form of infected, dried wheat galls, and represented an important opportunity to test our molecular methods for using small amounts of templates to generate libraries for high-throughput sequencing. The Anguina genus represents an important group of agricultural parasites that are resistant to long periods of desiccation. We have sequenced the nematodes and microbiomes from 8 infected galls, and are in the early stages of analyzing the data. The data set is already yielding early, interesting results including an estimate of genome size of approximately 229 million base pairs, a multi-partite mitochondrial genome, and an interesting microbiome which includes representatives of Bacillus, Marinomonas, and Wolbachia, which were identified initially using 16S amplicon sequencing, and later confirmed to be in the assemblies by BLAST algorithm. In the genome of Anguina tritici, we have identified a large cohort of single copy orthologous genes (BUSCO algorithm) which strengthens our preliminary assessment of these datasets. Furthermore, preliminary analyses have indicated that genes involved in trehalose expression (important in the desiccation resistance pathways) are present, but we have not yet investigated gene expression in this species. Change in knowledge: The development of a draft genome sequence for the nematode Anguina tritici and a refined protocol for sample preparation for next generation sequencing will add to the toolbox of researchers studying plant parasitic nematodes. Specifically for the metagenomics analysis, the new sample preparation protocol will enable better estimates of the abundance of associated microbes in a way that makes it possible to estimate representation in a way that can differentiate between amplification bias versus actual counts of bacterial genomes represented in the sample pool. Furthermore, we have developed a robust bioinformatics pipeline for analyzing genetic variation in populations of Anguina tritici and will test this pipeline on other populations of plant parasitic nematodes.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: During the summer of 2014, the data generated by the proposed research approach was incorporated into a Bioinformatics Bootcamp workshop (sponsored by NSF) targeting ~15 undergraduate students and ~5 faculty/staff from universities and colleges across the US. This data and the approaches developed are also part of our ongoing training/curriculum development as part of the NH-INBRE project (NIH). Other target audiences include students and researchers interested in understanding the genomics and microbiome associated with the genus Ditylenchus. Ultimately, this work will be of interest to the agricultural community and those interested in pest-management. Changes/Problems: High throughput sequencing is changing rapidly, and the instrumentation has changed to provide longer read lengths for the high throughput data. These changes have necessitated the need to rethink some of the approaches that have commonly been used in the past. What opportunities for training and professional development has the project provided? July 2014: Bioinformatics bootcamp--this NSF-sponsored workshop hosted 15 undergraduates from across the US, as well as several faculty. These students participated in learning to implement metagenomics analyses of the type and complexity proposed in this project. This project has provided the opportunity to engage graduate students from computer science in developing, refining and applying bioinformatics tools to the data. How have the results been disseminated to communities of interest? No results have yet been disseminated, but we are hoping to have data analyzed and ready to present in the upcoming year. What do you plan to do during the next reporting period to accomplish the goals? During the past year, we've been working on developing assays for generating data from a single Garlic Bloat nematode, Ditylenchus dipsaci, as well as using microbial 16S ribosomal sequences as barcodes to understand the microbial communities associated with individual specimens. We are working to obtain specimens from within the Ditylenchus genus to test these methods.
Impacts
What was accomplished under these goals?
The role of an organism in its environment is not simply determined by its own genetic blueprint but by its environment including the community of organisms in which it lives. In this project, we are testing the hypothesis that nematodes (roundworms) have unique microbial communities. The invasive garlic bloat worm is an important pest of Allium crops (leek, garlic, onion, etc.) We will be able to identify virtually all of the microbes associated with individuals of these species within and across populations. These observations will be the first critical step to understanding the role of the nematode microbiome in the life history of this important agricultural pest, their resilience to environmental change, and as potential targets for effective pest management. During the past year, we've worked on developing and refining protocols for generating high quality genomic reference sequences from individual nematodes and formalin-fixed specimens. We've also worked to implement metagenomics assays using a targeted portion of the 16S rRNA gene for determining the bacterial associates of this group of nematodes. We are currently working to develop collaborations with researchers working on this nematode, both in the US and abroad. Extensive work has been done within our group to refine the bioinformatics analysis pipelines associated with analyzing the high throughput sequencing data generated from this study.
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