Source: UNIVERSITY OF FLORIDA submitted to NRP
MITOCHONDRIAL METAGENOMICS (MTMG) AS A CRITICAL STEP TOWARDS ACCURATE AND EFFECTIVE DIAGNOSTICS OF PLANT-PARASITIC NEMATODE COMMUNITIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022041
Grant No.
2020-67013-30973
Cumulative Award Amt.
$466,000.00
Proposal No.
2019-08203
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2024
Grant Year
2020
Program Code
[A1181]- Tactical Sciences for Agricultural Biosecurity
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Entomology and Nematology
Non Technical Summary
The most effective strategy to management of any pest is the knowledge of pest identity. In nematode diagnostics, two critical gaps include: 1. the inability to adopt species-resolving mitochondrial gene markers for high-throughput sequencing (HTS) approaches (all species en mass rather than single individuals one at a time), and 2. the inability to ID sequenced individuals due to limited mitochondrial databases. The main goal of this project is to establish an HTS protocol for nematode diagnostics that provides accurate species and subspecies resolution, is broad in its taxonomic coverage, is expedient relative to low-throughput approaches, and reflects the entire genotypic composition of the plant-parasitic nematode community.In this project we propose to: 1. improve diagnostic capacity for plant-parasitic nematode species identification by developing, testing, and implementing a multi-locus system via shotgun sequencing of full mitochondrial genomes (Nema-mtMG); 2. develop a curated full mitochondrial genomes databse (Nema-MtDB) with all mitochondrial protein coding genes necessary to identify species that cannot be resolved with a single gene; 3. develop educational tools/workshops to facilitate training and adoption of the Nema-mtMG for plant-parasitic nematode diagnostics.To validate Nema-MtMG, our analyses will progress from known assemblages of nematode pest species to increasingly unknown in their species composition but involving taxa of agronomically important nematode clades. For Nema-mtDB construction, we will start with ~300 nematode species from cultures and follow up with species from soil samples. For Objectives 1 and 2, we will apply our recent protocols for mitochondrial DNA enrichment, Illumina HiSeq2500 sequencing, and phylogenetic-based bioinformatics.With the commitment of the USDA APHIS to "Safeguard American Agriculture" (Goal 2 of the Strategic Plan 2019-2023), our project is directly relevant to Objectives 2.1, 2.2 , and 2.6 that stress the role of innovative molecular diagnostics.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123130112050%
2120110104050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3130 - Nematodes; 0110 - Soil;

Field Of Science
1040 - Molecular biology; 1120 - Nematology;
Goals / Objectives
The main goal of this project is to establish a well-validated high-throughput protocol for nematode diagnostics that provides accurate species and subspecies resolution, is broad in its taxonomic coverage, is expedient relative to current low-throughput approaches, and reflects the entire genotypic composition of the entire plant-parasitic nematode community.We propose to address this goal via 3 objectives:Improve diagnostic capacity for plant-parasitic nematode species identification by developing, testing, and implementing the nematode mitochondrial metagenomics (Nema-mtMG) protocol.Develop a curated in-house nematode mitochondrial genomes database (Nema-mtDB) that includes all mitochondrial protein coding genes necessary to identify species that cannot be resolved with a single gene.Develop educational tools and workshops to facilitate training and adoption of the Nema-mtMG for plant-parasitic nematode diagnostics in a wide array of settings including academic research, biosecurity monitoring, and extension work.Objective 1. To ensure the accuracy and efficiency of the Nema-mtMG protocol, our analyses will proceed from known assemblages of nematode pest species (by assembling artificial plant-parasitic nematode communities from species maintained in cultures) to communities of increasing complexity in their species composition but involving known and unknown taxa of agronomically important nematode clades such as Heteroderidae, Longidoridae, Meloidogynidae, and Pratylenchidae. To properly test and validate the use of mtMG with Illumina HiSeq 2x250 bp pair-end sequencing for species-level diagnostics, we will compare it to the traditional approach that relies on the mitochondrial barcodes targeting the COI gene region. Data analyses will result in the development of new bioinformatics tools with error detection capability. Objective 2. Because mtMG sequences are only as good as reference databases that allow for annotation, we will develop a curated in-house Nema-mtDB that is publicly accessible. First, we will collate sequencing information from officially existing resources (e.g., BOLD, QBOL, Miduri, and NR-NCBI reference databases) and private collections of taxonomist colleagues and collaborators into a COI reference database compatible with HTS data processing and analysis. Importantly, we will extend beyond the COI database to include full mitochondrial genomes emphasizing all protein coding genes. To achieve this, we will apply HTS shotgun sequencing of mitochondrial genomes from individual plant-parasitic nematode species. Initially, their identity will be confirmed by traditional DNA tools and morphological analysis. The genomes will be assembled, with all protein coding genes annotated and assembled in the format necessary for HTS data analyses.Objective 3. To encourage the implementation of the Nema-mtMG we will produce protocol documents detailing all the steps of data handling, sample processing, HTS data generation, bioinformatic processing and reporting of results. We will produce presentations and webinars for online teaching, present seminars, and organize face-to-face workshops. These educational and outreach activities will be available to research laboratories, governmental agencies, and the broader scientific community. A linkage with similar efforts promoted by the BIOSCAN project of the International Barcode of Life members will hasten the development of the Nema-mtMG-based diagnostics.
Project Methods
Objective 1. To provide a mtMG tool, we will use an experimental design of scaling up from completely known to increasingly complex and unknown plant parasitic nematode communities. In Year 1, known communities will be assembled using nematode species from our ongoing cultures (33 species). Because detectability can be affected by the presence of different life stages and abundance, we will vary these variables for certain species. Since plant parasitic nematodes are always a part of a larger nematode community from which they must be differentiated, we will add taxa representing free-living nematode species including bacterial- (7 species) and fungal-feeders (5 species) that also are maintained in cultures.Each nematode species will be separately extracted, observed under a microscope, imaged, and hand-picked to assemble 8 types of mock communities each with 4 replicates for a total of 32 mock community samples. Prior to the assembly of mock communities, individual taxa will be ID-verified using Sanger sequencing with the COI gene. These sequences will also be used for mtDNA reference database construction (see Objective 2).Assembled mock communities will be processed for DNA extraction using DNeasy PowerSoil Kit. Equal DNA amounts (~1ng/µl) will be subsampled for library construction with the KAPA HyperPlus. We will apply animal-specific capture probes to enrich the gDNA pooled libraries for mitochondrial DNA (mtDNA) using SeqCap EZ Hyper Cap workflow. Enriched for mtDNA libraries will be sequenced on an Illumina HiSeq-2500 at the HCGS.Sequence data will be demultiplexed using the Illumina bclfasq Conversion Software and quality-filtered using FastQC. HiSeq adapters and low-quality bases will be trimmed using Trimmomatic v0.36 under default settings. Quality filtered and trimmed reads will be subjected to specifically developed bioinformatics pipeline (see below) and eventually assigned taxonomy against an in-house constructed mitochondrial nematode reference database (Nema-mtDB) consisting of all 13 PCGs (see Objective 2).Once validated on mock communities, we will scale up to more complex samples from agricultural fields in Year 2-3. We will start with soil samples that have been repeatedly evaluated by two nematode diagnostic labs (at the University of Nebraska, Lincoln and the University of Florida, Gainesville). These fields provide realistic examples of mixed populations of important pest species and include different cropping systems: a corn-soybean rotation in the Midwest and a peanut-corn rotation in Florida. Specifically, we will collect soil samples from: a corn-soybean rotation in the Midwest (2 different field sites x 6 reps = 12 samples), a soybean-cotton system in Arkansas (2 different field sites x 6 reps = 12 samples), a peanut-cotton rotation in Florida (2 different field sites x 6 reps = 12 samples) for a total of 36 environmental communities that we have previously worked with. Since many pest species have wide-world distributions, in collaboration with Dr. Hodda of CSIRO, Australia, we will test for geographic consistency by processing 36 soil samples from Australia that contain the same Heterodera, Meloidogyne, and Pratylenchus species observed in US soils.Objective 2. We will build a multigene nematode reference database - Nema-mtDB by: 1. retrieving all COI nematode reference from the curated Midori database; 2. retrieving all COI nematode references sequences from the QBOL database focusing on DNA barcoding of quarantine pests including nematodes; 3. adding personal collections of specimens that we identified in our past work; 4. adding COI Sanger-sequenced individual specimens from samples that we are currently working on; 5. adding all plant-parasitic species that we have selected for this project.Header sequences will be reformatted to include consistent NCBI taxonomic lineages and gene annotations. To allow for phylogenetic-based placement and taxonomic classification of the sequencing reads, we will construct first gene alignments at the family level using Clustal Omega. These alignments were used to construct Maximum Likelihood trees using RAXML.To ensure full database functionality, will rapidly expand to PCGs by generating full mitochondrial genomes (with mtMG protocol). We will apply protocol for species present in communities in this project, but also continue the expansion by using species from other projects.To assemble mitochondrial genomes (and all PCGs) of all selected individual plant-parasitic nematode specimens, reads will be first demultiplexed, quality-filtered, and trimmed as described above. Trimmed reads will be aligned to taxonomically broad reference FASTA in protein space (to speed up the process) with BWA-mem (Li 2013) to assign phylum level taxonomic classification and all non-Nematoda reads will be filtered out. Retained Nematoda reads will be subjected to mitochondrial baiting and iterative mapping (MITObim) approach to reconstruct complete mitochondrial genomes of all individual species. Initially, we will use Sanger-generated COI reference sequences to guide (bait) seed reads, but as Nema-mtDB becomes populated with full mitochondrial genomes, we will use these reference genomes instead. We propose to assemble reads into mito-scaffolds using SOAPdenovo bioinformatics tools and concatenate them using TGICL. Mitochondrial sequences will be annotated using the MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py). Protein-coding gene annotations will be manually curated to ensure the correct start and stop codons. Each of the 13 PCGs will be extracted from the mito-scaffolds, added to respective gene/family fasta, and aligned by Clustal Omega as described above.As the process of alignments and tree construction will be frequently repeated at the early stages of this project, the database (with alignments and trees) will be hosted and available for download from Porazinska's server. However, long-term storage will be arranged via BOLD.In addition to expanding the Nema-MtDB with Sanger- and Illumina-generated reference sequences from this project, we will also collate available yet unpublished sequences from our taxonomist collaborators.Objective 3. The mtMG approach to nematode diagnostics should outperform current tools in many respects (e.g., high precision and accuracy, high throughput, discovery of new species). Since diagnostic labs still strongly rely on morphology or low-throughput molecular tools, the transition to Nema-mtMG will require substantial investment in training and outreach activities. We are currently organizing a workshop: "NemataBOL for Nematode Barcode of Life" for the International Congress of Nematology Societies in Antibes, France in 2020 to develop a multicollaborative effort to effectively transition nematode diagnostics to metabarcoding tools.In order to help the transition, we will develop effective teaching and training materials that first will be tested in two nematode diagnostic labs (UNL and UFL) to refine all developed processing manuals and training materials (e.g., videos and PPP). We will use all these resources in organized workshops initially conducted at the national meetings of the Society of Nematologists and American Phytopathological Society. To allow for self-paced training, all resources will be hosted and shared via a GitHub page.

Progress 05/01/20 to 04/10/24

Outputs
Target Audience:Targeted intended audience included scientific colleagues and professionals at national meeting of the Society of Nematologists at Gulf Shores, AL in 2021 andColumbus, OH in 2023. We also targeted the International Congress of Nematologists in Antibes, France in 2022.Altogether, we presented 6 session talks, 1 poster, and 1 workshop.In addition, the audience included students, postdocs and faculty of the Entomology and Nematology Department at the University of Florida, Gainesville, FL andDepartment of Agroecology and Entomology and Plant Pathology. Aarhus University, Denmarkwhere presented 5 invited seminars as part of departmental seminar series. Moreover, we are scheduled to deliver 1 plenary talk as well as 1 workshop at the upcoming Society of Nematologists at Park City, UT in 2024. Talks/Posters: Gendron E), JL Sevigny, TO Powers), WK Thomas, I Byiringiro, DL Porazinska. Utilizing shotgun mitochondrial metagenomics for improved data collection and identification of nematodes. National Meeting of theSociety of Nematologists, Gulf Shores, AL, 2021. Borgmeier A, K Gattoni, T Harris,P Mullin, DL Porazinska, K Powers, TO Powers. Nuance or numbers: taxonomic bias in approaches to measure nematode biodiversity. National Meeting of theSociety of Nematologists, Gulf Shores, AL, 2021. Gendron E, JL Sevigny, I Byiringiro, TO Powers, WK Thomas, DL Porazinska. 2022. Development of mitogenomic protocols for the enhancement of nematode identification and biodiversity studies. Talk and poster, ICN, Antibes, France. Porazinska DL,Gendron E,JLS Sevigny, TO Powers, WK Thomas. 2022. Molecular approaches for evaluation of nematode community diversity: paving a way into the future. Invited talk.ICN, Antibes, France Powers TO, DL Porazinska, B Adamas, A Borgmeier,R Higgins, T Harris, P Mullin, K Powers. 2022. DNA barcoding of individual specimen using COI and 18S in nematode community analyses: advantages of a combined approach.ICN, Antibes, France. Porazinska DL,Gendron EMS,X Qing, JL Sevigny, H Li, Z Liu, E King, M Blaxter, TO Powers, WK Thomas. 2023. Comparative mitochondrial genomics in Nematoda: astonishing variation in compositional strand biases and mutation rates driven more by life traits than phylogeny. Society of Nematologists, Columbus, OH. Porazinska DL, K Powers, P Mullin, TO Powers. Response of nematode communities to alkalinity and climate warming. 2024. SON, Park City, UT. Workshops Powers TO, DL Porazinska. 2022. DNA Barcoding.ICN, Antibes, France. Gendron E, J Sevigny, DL Porazinska. 2024. Mitochondrial metagenomics for high throughput nematode identification - What is it? How good is it? How to use it? SON. Park City, UT Seminars Porazinska DL. 2021. Soil community ecology through an eye of a nematode. Entomology and Nematology Department. University of Florida, Gainesville, FL. Gendron E. 2021. Shotgun mitochondrial metagenomics for improved nematode biodiversity studies. Entomology and Nematology Department. University of Florida, Gainesville, FL. Gendron E, JL Sevigny, I Byiringiro, TO Powers, WK Thomas, DL Porazinska.2022.Nematode mitochondrial metagenomics: benefits and risks. Entomology and Nematology Department.University of Florida, Gainesville, FL. Gattoni K, E Gendron, JP McQueen, R Shepherd, DL Porazinska.2022. Nematode biodiversity: why it is important and how we study it.Entomology and Nematology Department.University of Florida, Gainesville, FL. Porazinska DL. 2022. Nematodes as models for studies of community assembly. Department of Agroecology and Entomology and Plant Pathology. Aarhus University, Denmark. Changes/Problems:Due to Covid, we were unable to travel to Australia for collection of agricultural samples, however we substituded these samples with samples collected during other research opportunites representibg awider array oflocations both international (Botswana, Canada, Ireland, Norway, Poland, Rwanda) andnational (Colorado, Missouri, North Carolina, Puerto Rico, Tennessee, Texas). What opportunities for training and professional development has the project provided?Overall, the project has provided for training of 11 students (7 BS, 4 MS, 6 PhD) and3 postdocs.The scope of work and engagement included lab activities (such as nematode culturing, extractions, nematode isolation, DNA extractions), computational activities (sequencing data analyses), and dissemiation of results (talks and seminars at national meetings and departmental seminar series, workshops and code atnational meetings and github, and publications). How have the results been disseminated to communities of interest?We presented talks and seminars, workshops, and published: 7 talks and 1 posterat the Society of Nematologists 2021 and 2023 and International Congress of Nematologists, 2022 5 invited seminars at departmental seminar series 2 workshops 3 publications What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Overall, we accomplished all objectives. In lieu of Objective 1, we analyzed a total of 32 and 65 environmental samples. As intended, we scaled upfrom mock communities with known nematode species to agricultural samples from strawberry fields in Florida that are characterized by simplified nematode foodwebs and several well-characterized plant parasites allowing for careful validation of our methods. We have also sequenced and started analyzing more complex environmental samples from all over the world. At this point, the potential for mtMG is high, but only if the reference sequences of closely related taxa are present in the mtMG database (mock community recovery of almost all species vs. large disparity of detected taxa between morphology and mtMG for agricultural samples). Because the database is very limited and experiences a slow progress in its expansion for new genomes across the entire Nematoda tree, the mtMG approach might be limited. In lieu of Objective 2, we created and curated a mitochondrial reference database (Nema_mtDB) by collating sequencing information from officially existing resources (e.g., BOLD, QBOL, Miduri, and NR-NCBI, private collections), identifying erroneous sequences, standardizing gene names, and updating all IDs with the currently accepted nematode taxonomy. We also expanded the databases by adding approximately 100 novel mitochondrial genomes as well as 1000 COX1 sequences. Finally, in lieu of Objective 3, we disseminated all information via publications, presentations, and sharing all code/scripts for all the steps of processing the data and databases construction (GitHubhttps://github.com/Joseph7e/NG-MMAT ).Additionally, we organized workshops (1 at the ICN 2022 in France and one at the SON 2024 Utah) at scientificmeetings.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Gendron EMS, X Qing, JL Sevigny, H Li, Z Liu, E King, M Blaxter, TO Powers, WK Thomas, DL Porazinska. Comparative mitochondrial genomics in Nematoda: astonishing variation in compositional strand biases and mutation rates driven more by life traits than phylogeny. BMC Genomics.
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Gendron EMS, CJ Oliveira, J Desaeger, DL Porazinska. Improving understanding of nematode communities in agricultural settings: comparison of mitometagenomics and morphology. Metabarcoding and Metagenomics.


Progress 05/01/22 to 04/30/23

Outputs
Target Audience: Targeted intended audience included scientific colleagues and professionals at the 2022 International Congress of Nematologists in Antibes, France, where we presented 3 session talks, 1 poster, and 1 workshop. In addition, the audience included students, postdocs and faculty of the Entomology and Nematology Department at the University of Florida, Gainesville, FL, where we presented 1 invited seminar as part of departmental seminar series. Changes/Problems:Due to Covid we were unable to travel to Australia for collection of agricultural samples, however we were able to substitute these efforts with collected samples from a wider array of environmental environments and locations both international (Botswana, Canada, Ireland, Norway, Poland, Rwanda) as well as national (Colorado, Missouri, North Carolina, Puerto Rico, Tennessee, Texas). What opportunities for training and professional development has the project provided? During the last year, the project far has provided for training and work of 3 students (1BS, 2MS, 1 PhD) and 3 postdocs. The scope of work and engagement included lab activities (such as nematode culturing, extractions, nematode isolation, DNA extractions, and sequencing data analyses) but also presentation of talks and seminars at national meetings and departmental seminar series, and leading publications in high impact journals. How have the results been disseminated to communities of interest? We presented talks and seminars: 6 talks and 1 poster at the International Congress of Nematologists, Antibes, France, 2022 2 invited seminars at departmental seminar series of the Entomology and Nematology Department of the University of Florida, FL, 2022 3 publications What do you plan to do during the next reporting period to accomplish the goals? Gendron EM, Q Xue, H Li, Z Liu, JL Sevigny, E King, M Blaxter, TO Powers, WK Thomas, DL Porazinska. Comparative mitochondrial genomics in Nematoda: astonishing variation in compositional strand biases and mutation rates driven more by life traits than phylogeny. @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}@font-face { panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536859905 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}p.MsoCommentText, li.MsoCommentText, div.MsoCommentText {mso-style-noshow:yes; mso-style-priority:99; mso-style-link:"Comment Text Char"; margin:0in; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}span.MsoCommentReference {mso-style-noshow:yes; mso-style-priority:99; mso-ansi-font-size:8.0pt; mso-bidi-font-size:8.0pt;}p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpFirst, li.MsoListParagraphCxSpFirst, div.MsoListParagraphCxSpFirst {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpMiddle, li.MsoListParagraphCxSpMiddle, div.MsoListParagraphCxSpMiddle {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpLast, li.MsoListParagraphCxSpLast, div.MsoListParagraphCxSpLast {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}span.CommentTextChar {mso-style-name:"Comment Text Char"; mso-style-noshow:yes; mso-style-priority:99; mso-style-unhide:no; mso-style-locked:yes; mso-style-link:"Comment Text"; mso-ansi-; mso-bidi-;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;}ol {margin-bottom:0in;}ul {margin-bottom:0in;}

Impacts
What was accomplished under these goals? We have provided all code/scripts for all the steps of processing the data and databases construction for access to the public. These code scripts are on our GitHubhttps://github.com/Joseph7e/NG-MMAT . Additionally, we started planning a training workshop for the next year SON meeting. @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}@font-face { panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536859905 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}p.MsoCommentText, li.MsoCommentText, div.MsoCommentText {mso-style-noshow:yes; mso-style-priority:99; mso-style-link:"Comment Text Char"; margin:0in; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}span.MsoCommentReference {mso-style-noshow:yes; mso-style-priority:99; mso-ansi-font-size:8.0pt; mso-bidi-font-size:8.0pt;}p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpFirst, li.MsoListParagraphCxSpFirst, div.MsoListParagraphCxSpFirst {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpMiddle, li.MsoListParagraphCxSpMiddle, div.MsoListParagraphCxSpMiddle {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}p.MsoListParagraphCxSpLast, li.MsoListParagraphCxSpLast, div.MsoListParagraphCxSpLast {mso-style-priority:34; mso-style-unhide:no; mso-style-qformat:yes; mso-style-type:export-only; margin-top:0in; margin-right:0in; margin-bottom:0in; margin-left:.5in; mso-add-space:auto; mso-pagination:widow-orphan; ; mso-fareast-}span.CommentTextChar {mso-style-name:"Comment Text Char"; mso-style-noshow:yes; mso-style-priority:99; mso-style-unhide:no; mso-style-locked:yes; mso-style-link:"Comment Text"; mso-ansi-; mso-bidi-;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;}ol {margin-bottom:0in;}ul {margin-bottom:0in;}

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 1. Gendron E, JL Sevigny, I Byiringiro, TO Powers, WK Thomas, DL Porazinska. 2022. Development of mitogenomic protocols for the enhancement of nematode identification and biodiversity studies. Talk and poster, ICN, Antibes, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 2. Porazinska DL, Gendron E, JLS Sevigny, TO Powers, WK Thomas. 2022. Molecular approaches for evaluation of nematode community diversity: paving a way into the future. Invited talk. ICN, Antibes, France
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 3. Powers TO, DL Porazinska, B Adamas, A Borgmeier, R Higgins, T Harris, P Mullin, K Powers. 2022. DNA barcoding of individual specimen using COI and 18S in nematode community analyses: advantages of a combined approach. ICN, Antibes, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 4. Powers TO, DL Porazinska. DNA Barcoding. Workshop. ICN, Antibes, France 5. Gendron E, JL Sevigny, I Byiringiro, TO Powers, WK Thomas, DL Porazinska. 2022. Nematode mitochondrial metagenomics: benefits and risks. Entomology and Nematology Seminar Series.
  • Type: Other Status: Other Year Published: 2022 Citation: Gattoni K, E Gendron, JP McQueen, R Shepherd, DL Porazinska. 2022. Nematode biodiversity: why it is important and how we study it. Nematology Seminar Series.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 1. Gattoni K, E Gendron, JP McQueen, R Shepherd, R Sandoval Ruiz, L Uy, DL Porazinska. 2023. Curated 18S nematode database (18S-Nema-DB) for improved metabarcoding analyses. Journal of Nematology. https://doi.org/10.2478/jofnem-2023-0006
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Gendron E, JL Sevigny, I Byiringiro, TO Powers, WK Thomas, DL Porazinska. 2023. Nematode mitochondrial metagenomics  a new tool for biodiversity studies. Molecular Ecology Resources. https://doi.org/10.1111/1755-0998.13761
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 3. Borgmeier A, T Harris, R Higgins, K Gattoni, P Mullin, DL Porazinska, K Powers, TO Powers. 2022. Plectus of the prairie: A case study of taxonomic resolution from a nematode biodiversity survey. Journal of Nematology. e2022-1, 54: https://doi.org/10.2478/jofnem-2022-0039
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Ren Y, DL Porazinska, Q Ma, S Liu, H Li, Q Xue. A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling. Submitted. Applied Soil Ecology.
  • Type: Other Status: Published Year Published: 2023 Citation: Publication of Nema-mtDB: https://github.com/WormsEtAl/Nematode-Mitochondrial-Database
  • Type: Other Status: Published Year Published: 2022 Citation: Publication of 18S-Nema-DB: https://github.com/WormsEtAl/18SNemaBase


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:Targeted intended audience included scientific colleagues and professionals at the national meeting of Society of Nematology at Gulf Shores, AL, 2021, where we presented the session talks listed below. In addition, the audience included students, postdocs and faculty of the Entomology and Nematology Department at the University of Florida, Gainesville, FL, where we presented at 2 invited seminars as part of departmental seminar series. SON presentations: Gendron E), JL Sevigny, TO Powers), WK Thomas, I Byiringiro, DL Porazinska. Utilizing shotgun mitochondrial metagenomics for improved data collection and identification of nematodes. National Meeting of the Society of Nematologists, Gulf Shores, AL, 2021. Borgmeier A, K Gattoni, T Harris, P Mullin, DL Porazinska, K Powers, TO Powers. Nuance or numbers: taxonomic bias in approaches to measure nematode biodiversity. National Meeting of the Society of Nematologists, Gulf Shores, AL, 2021. ENY seminars: Porazinska DL. Soil community ecology through an eye of a nematode. Spring 2021. https://entnemdept.ufl.edu/seminar/recordings/Dorota_Porazinska_seminar.mp4 Gendron E. Shotgun mitochondrial metagenomics for improved nematode biodiversity studies Fall 2021. https://entnemdept.ufl.edu/seminar/recordings/Eli_Gendron_Seminar.mp4 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the last year, the project far has provided for training and work of xx students (4BS, 1 MS, 2 PhD) and 2 postdocs. The scope of work and engagement included lab activities (such as nematode culturing, extractions, nematode isolation, DNA extractions, and sequencing data analyses) but also presentation of talks and seminars at national meetings and departmental seminar series. How have the results been disseminated to communities of interest? We presented talks and seminars: 2 talks at the national meeting of the Society of Nematologists, Gulf Shores, AL, 2021 2 invited seminars at departmental seminar series of the Entomology and Nematology Department of the University of Florida, FL, Spring and Fall 2021 What do you plan to do during the next reporting period to accomplish the goals?1. Analyze and validate environmental data from known agricultural settings that provide realistic examples of mixed populations of important pest species in Florida. 2. Analyze and validate environmental data from unknown settings likely supporting new and diverse nematode species. 3. Continue collecting specimens of differentnematode species and process these for COI using Sanger sequencing and formitochondrial genome sequencing using high throughput sequencing. 4. Continue expanding the database for genomes. 5. Attempt to assess usefulness of mitochondrial genomes in better understanding of nematode phylogeny. 6. Participate in the International Congress of Nematology scheduled for May 2022, France for three main activities: a workshop, 1 session talk, and a symposium talk, and a poster for which four abstracts were submitted and accepted. 7. Participate in the national meeting of the Society of Nematologists scheduled for Sept 2022, Alaska for two main activities: 3 session talks and a training workshop. 8. Expend training tutorials. 9. Convert accumulated materials to educational materials including manuals and videos. 10. Publish two manuscripts, Mitochondrial metagenomics for analyses of nematode diversity with projected date of submission of March 31, 2022 to Molecular Ecology Resources Validation of mtMG approach on environmental samples with a projected date July 31, 2022.

Impacts
What was accomplished under these goals? In lieu of fulfilling Objective 1, We sequenced and analyzed the 32 mock communities and evaluated them accurate recovery of expected nematode species. We are excited to report that the mtMG approach is highly effective: Out of 30 nematode species that were used to assemble these communities, only three (Ditylenchus halictus, Belonolaimus longicaudatus, and Pratylenchus hippeastri) were not detected. Nematode species were detected across all communities even those were composed of single nematode eggs per each species. Closely related species within families of Bursaphelenchidae, Heteroderidae, Meloidogynidae, and Pratylenchidae were differentiated and detected. Although the detection was reinforced/confirmed by recovery of species across multiple PCGs, some genes provided a better detection (i.e., CO1 and NAD5) than others (e.g., NAD1-NAD4). The false positive errors did not appear to be a major problem, but we are still in the processes of formal evaluation of these errors as well of identifying potential bioinformatic solutions to correct these errors. Metagenomic data also allowed for the assembly of 59 mt genomes, crucial to the continuing process of expanding the database. In addition to the mock communities, we sequenced a total of ~150 environmental samples. As intended, we first scaled up to local agricultural samples from strawberry fields in Florida that are characterized by simplified nematode foodwebs and several well-characterized plant parasites allowing for careful validation our methods. However, given the successful results from the mock communities, we have also attempted sequencing of ~80 complex, but not well-characterized nematode communities. Both sets of samples are scheduled for bioinfomatic processing and analyses in upcoming months. In lieu of fulfilling Objective 2, We assembled, annotated, and curated 59 mitochondrial genomes. At this point, the added genomes to the database consists of 46 complete mitochondrial genomes and 13 mostly complete genomes. The extent of coverage varies across all 13 PCGs. We evaluated the success of genome assemblies based on number of individuals, DNA concentration, AT/GC content, and taxonomy. Although AT/GC content (assembly success declines as AT/GC content goes up), all other factors are also important, but not in a clearly predictable manner. As we sequence and assemble more genomes, these patterns will become clearer. All newly sequenced genomes are validated via assignment statistic indices and tree building. We have observed few false positive errors. To improve capture of nematode-derived mtDNA and lower the risk of DNA from other organisms, redesigned capture probes to enrich the gDNA pooled libraries for mitochondrial DNA (mtDNA). We are in the process of testing the new set of probes and data for these tests will be available by the end of February 2022. Finally, to keep expanding the COI database, newly identified nematodes species from environmental samples have been isolated, photographed, morphologically identified and Sanger sequenced for COI. In lieu of fulfilling objective 3, We have started to organize and streamline all code/scripts for all the steps of processing the data and databases construction for access to the public. These code scripts will be continually updated on GitHubhttps://github.com/Joseph7e/NG-MMAT

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Gendron E, JL Sevigny, TO Powers, WK Thomas, I Byiringiro, DL Porazinska. 2021. Utilizing shotgun mitochondrial metagenomics for improved data collection and identification of nematodes. Journal of Nematology. Vol. 53, pp. 10-11.
  • Type: Other Status: Published Year Published: 2021 Citation: Borgmeier A, K Gattoni, T Harris, P Mullin, DL Porazinska, K Powers, TO Powers. 2021. Nuance or numbers: taxonomic bias in approaches to measure nematode biodiversity. Journal of Nematology. Vol. 53, pp. 4-5.
  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Borgmeier A. Nematode Biodiversity in Lincoln Nebraskas Tallgrass Prairie Corridor. University of Nebraska August, 2021 M.S. Thesis


Progress 05/01/20 to 04/30/21

Outputs
Target Audience: Targeted intended audience was the International Congress of Nematology scheduled for May 2020, France, however due to Covid-19 pandemic the ICN has been postponed to May 2022. The proposed activities included a workshop, a session talk, a symposium talk, and a poster for which four abstracts were submitted and accepted. Powers TO, DL Porazinska. DNA barcoding. Workshop. T Powers, DL Porazinska, B Adams, B Butera, R Higgins, T Harris, P Mullin, K Powers. DNA barcoding using COI in nematode community analyses: advantages and limitations. DL Porazinska, B Butera, TO Powers, JL Sevigny, WK Thomas. Molecular approaches for evaluation of nematode community diversity: paving a way into the future. E Gendron, TO Powers, JL Sevigny, WK Thomas, DL Porazinska. Using shotgun metagenomics to expand the diversity of the mitochondrial genome data for nematodes. Changes/Problems:Genome assembly from samples containing too few individuals (<15) appears to occasionally be associated with inability to assemble complete genomes. A similar problem might be associated with samples that contain too manyindividuals (>2000) likelydue to hyper intragenomic variation. We are in the process of formally evaluating these issues. What opportunities for training and professional development has the project provided?The project thus far has provided for training and work of 5 students (2 BS, 1 MS, 3 PhD) and a 1 postdoc. The scope of work and engagement included lab activities such as nematode culturing, extractions, nematode isolation, DNA extractions, and sequencing, but also sequencing data analyses and development of new bioinformatic tools. How have the results been disseminated to communities of interest? The intended method of dissemination were scientific presentations at the International Congress of Nematology scheduled for May 2020, France, however due to Covid-19 pandemic the ICN has been postponed to May 2022. The proposed activities included a workshop, a session talk, a symposium talk, and a poster for which four abstracts were submitted and accepted. Powers TO, DL Porazinska. DNA barcoding. Workshop. T Powers, DL Porazinska, B Adams, B Butera, R Higgins, T Harris, P Mullin, K Powers. DNA barcoding using COI in nematode community analyses: advantages and limitations. DL Porazinska, B Butera, TO Powers, JL Sevigny, WK Thomas. Molecular approaches for evaluation of nematode community diversity: paving a way into the future. E Gendron, TO Powers, JL Sevigny, WK Thomas, DL Porazinska. Using shotgun metagenomics to expand the diversity of the mitochondrial genome data for nematodes. What do you plan to do during the next reporting period to accomplish the goals? The major goal of the upcoming reporting period is completion of the methodology and protocols for nematode species identification using mitochondrial metagenomics.The methods (from processing of samples for DNA sequencing to processing the sequencing data) will be thoroughly tested on nematode mock communitiesto allow for evaluation of all potential errors as well as parametrization of the bioinformatics. Assess the mitochondrial genome sequencing success rate to develop best practices protocols. Continue collecting specimens of differentnematode species and process these for COI using Sanger sequencing and formitochondrial genome sequencing using high throughput sequencing. Continue expanding the database and refining bioinformatics for genome assembly, annotation, and tree building. Complete analyses of mock communities to evaluate methodological accuracy and sensitivity. Once validated on mock community, collect field samples that have been repeatedly evaluated. These fields provide realistic examples of mixed populations of important pest species in the Midwest and in Florida. Begin to develop educational materials including manuals and videos. Participate in scientific meetings to present results from Year1. Publish two manuscripts - methods for mitochondrial genome sequencing and database development and bioinformatics tools and pipeline for data analyses.

Impacts
What was accomplished under these goals? In lieu of fulfilling Objective 2 (develop a curated in-house Nema-mtDB), we have collated sequencing information from officially existing resources including BOLD, QBOL, Miduri, and NR-NCBI reference databases, but also private collections, and generated in this project genomes and COI sequences into a mitochondrial database that includes all mitochondrial protein coding genes (PCGs). At this point, the database consists of ~300 complete mitochondrial genomes (~100 sequenced via this project last year). Predictably, COI gene provides the highest coverage (~7000 COI non-redundant sequences that are representative of ~1,000 species). In addition to collating available resources, the taxonomic strings that accompanied the reference sequences have been bioinformatically and manually curated to conform to the most current nematode taxonomic information based on the WoRMs database. This curation/standardization was required to allow for database compatibility with high throughput sequencing analyses, and to improve the ease of access to phylogenetic and trophic information within the database. To allow for phylogenetic analyses, we have started working toward generation of first phylogenetic trees at the family level as well as bioinformatic framework for their expedient expansion. These trees are crucial for "species" delimitation and identification. We sequenced 74 new full mitochondrial genomes of nematode species that we had in cultures (see Other Products - Data). The species represented mostly plant parasitic nematodes, but also expanded to other taxa across the entire Nematoda Tree of Life. For each species, two separate datasets were generated: 1.) Whole-genome shotgun sequencing (WGS) datasets using a standard library preparation method and skim (low coverage) sequencing and 2.) WGS with mtDNA enriched using hybridization capture probes and skim sequencing. Assembled genomes and annotated genes. Of the 74 species, we were able to recover complete mitochondrial genomes for 36 species (50% overall success rate), and complete COX1 sequences from additional 22 species (78% overall success rate). Preliminary observations suggest that samples that failed to produce mtDNA genomes were often associated with either samples of too low DNA concentrations (below detection limit by Qubit) from too few (15) nematode individuals, or from too high DNA concentrations, too many (>1500) nematode individual nematodes. Species from the failed samples were re-processed and re-sequenced. Moreover, additional 20 recently acquired species were also sequenced. Data is currently being analyzed. Assembled artificial plant-parasitic communities from known cultures and processed them for mitochondrial enrichment, and sequencing. Specifically, each nematode species (10 plant parasitic, 7 bacterial feeders, 5 fungal feeders) was separately extracted, observed under a microscope, and hand-picked to assemble 8 types of mock communities each with 4 replicates for a total of 32 mock community samples. Assembled mock communities were processed for DNA extraction using DNeasy PowerSoil Kit. Equal DNA amounts (~1ng/µl) was subsampled for library construction with the KAPA HyperPlus and spiked with C. elegans. We applied animal-specific capture probes to enrich the gDNA pooled libraries for mitochondrial DNA (mtDNA) using SeqCap EZ Hyper Cap workflow. Enriched for mtDNA libraries were sequenced on an Illumina HiSeq-2500 at the HCGS. The data is in the process of analysis. Developed code/scripts for all the steps of processing the data and databases construction. First pass code scripts are hosted on GitHubhttps://github.com/Joseph7e/NG-MMAT. To keep expanding the COI database, 560 newly identified nematodes species from environmental samples have been isolated, photographed, morphologically identified and Sanger sequenced for COI.

Publications