Charakterisierung und Sequenzanalyse von T-DNA Insertionsstrukturen und paralogen Bereichen im Genom von Arabidopsis thaliana basierend auf Optimierungen der Daten-Erfassung und -Auswertung fur die GABI-Kat-Kollektion

Kleinbölting N. Charakterisierung und Sequenzanalyse von T-DNA Insertionsstrukturen und paralogen Bereichen im Genom von Arabidopsis thaliana basierend auf Optimierungen der Daten-Erfassung und -Auswertung fur die GABI-Kat-Kollektion. Bielefeld: Universitätsbibliothek; 2015.Weist das Genom eines Organismus einen Defekt in einem bestimmten Gen auf, der dessen Funktion komplett aufhebt, spricht man von einer sogenannten Knockout-Mutante. Diese sind ein unentbehrliches Werkzeug für die reverse Genetik, denn aus der Beobachtung der Mutante lassen sich Rückschlüsse auf die Funktion des defekten Gens ziehen. Die GABI-Kat-Kollektion wurde zu dem Zweck entwickelt, Zugriff auf knapp 72.000 potentiell interessante Knockout-Linien in Arabidopsis thaliana anzubieten. Zur Generierung der Linien wurde die durch Agrobacterium tumefaciens vermittelte Transformation verwendet, bei der ein Teil des Tumor-induzierenden (Ti)-Plasmids, die T-DNA, in das Genom des Wirts-Organismus an einer zufälligen Stelle stabil integriert wird und aufgrund ihrer Größe zum vollständigen Knockout eines dort kodierten Gens führen kann. Ein großer Vorteil gegenüber anderen Methoden zur Generierung von Knockouts ist, dass die Sequenz der integrierten T-DNA bekannt ist. So lassen sich Primer erstellen, mit denen Flanking Sequence Tags (FSTs) sequenziert werden können, die zur Insertion benachbarte Sequenzen beinhalten. Durch Analyse der Sequenzierergebnisse lassen sich Rückschlüsse auf den genauen Ort der Insertionen ziehen. Forscher können eine GABI-Kat Linie bestellen, die eine für sie interessante Insertion enthält. Daraufhin wird über eine klärende PCR und nachfolgende Sequenzierung die Anwesenheit der Insertion bestätigt und die Samen schließlich an den Besteller versendet. Im Rahmen dieser Arbeit wurden verschiedene Methoden innerhalb des GABI-Kat Projektes etabliert, um die Anzahl der bestätigten Insertionen zu erhöhen. Diese Methoden umfassten eine exaktere Vorhersage der FST-basierten Insertionsposition, die Einführung von Kontaminationsgruppen, um die richtige Linie unter identischen Vorhersagen in verschiedenen Linien zu identifizieren und die Aufklärung von schwerwiegenden Fehlern in der Zuordnung von FSTs. Außerdem wurden zwei Methoden zur Steigerung der Zuverlässigkeit von Analysen in paralogen Bereichen des Genoms entwickelt. Dies umfasste zum Einen paraloge Gruppen zur Aufklärung von Mehrdeutigkeiten in den Insertions-Vorhersagen und zum Anderen die Entwicklung eines Werkzeugs zur Generierung von eindeutigen Primern im Genom von A. thaliana. Diese Maßnahmen führten zu einem signifikanten Anstieg der Bestätigungsrate, die angibt, wie viele Insertionen experimentell bestätigt werden konnten von 78 % auf fast 88 %. Sie trugen maßgeblich dazu bei, dass möglichst viele Forscher die für sie interessanten Linien erhalten und unterstützten damit den wissenschaftlichen Informationsgewinn. Aufgrund von Duplikationen und daraus resultierenden paralogen Genen im Genom von Arabidopsis thaliana führen deren Knockouts in vielen Fällen nicht zwangsläufig zu einem sichtbaren Phänotyp. Durch mindestens eine zweite intakte Kopie, die die Funktion des defekten Gens teilweise oder komplett übernehmen kann, bleiben mögliche Auswirkungen des Knockouts oft unbemerkt. Um dieses Problem zu adressieren, wurde in dieser Arbeit eine Liste homologer Gene in A. thaliana berechnet anhand derer überprüft werden kann, ob der Knockout mehrerer Gene bei der Untersuchung eines Gens erforderlich ist. Anhand dieser Liste wurden 200 Doppelmutanten für vielversprechende Kandidaten-Paare durch Kreuzungen generiert. Bei der visuellen Analyse dieser Doppelmutanten zeigte sich eine deutliche Zunahme der beobachtbaren Phänotypen gegenüber Einzelmutanten. Die Informationen zu den Doppelmutanten und die Liste homologer Gene sind über eine im Internet verfügbare Benutzerschnittstelle verfügbar, die im Rahmen dieser Arbeit entwickelt wurde. Auf Basis einer großen Anzahl von Sequenzen, die aus der Bestätigung einzelner Insertionen in GABI-Kat stammen, konnten gemeinsame Charakteristika von T-DNA Insertionen aufgezeigt werden. Die Integration der T-DNA führt fast immer zu einer Veränderung der Ursprungssequenz in Form von kleineren Deletionen (ca. 1-50 bp) und kleineren Duplikationen (ca. 1-10 bp). In seltenen Fällen wurden auch Inversionen und große Deletionen von mehreren Kilobasen beobachtet. Am Übergang zwischen T-DNA und Kerngenom von A. thaliana finden sich normalerweise kürzere Bereiche von Mikrohomologie, die in Einzelfällen bis zu 20 bp groß sind, oder ein Filler, dessen Ursprung in den meisten Fällen im Genom von A. thaliana zu finden ist und der wiederum oft kurze Bereiche von Mikrohomologie zur jeweils benachbarten Sequenz aufweist. Des Weiteren ist die T-DNA häufig um einige Basen bezüglich der erwarteten Schnittstelle in der Border verkürzt. Alle diese Eigenschaften einer T-DNA Insertion deuten darauf hin, dass die Integration der T-DNA hauptsächlich mit Hilfe des Reparaturmechanismus NHEJ stattfindet. Aber auch die Beteiligung anderer Reparaturmechanismen wie MMEJ und SDSA ist wahrscheinlich. Diese Annahmen konnten in dieser Arbeit erstmals mit einer großen Fallzahl untersucht und belegt werden

Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities

Schenk J, Kleinbölting N, Traunspurger W. Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities. Ecology and Evolution. 2020;10(6): ece3.6104.Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single-specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis.

BACKGROUND: Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. RESULTS: We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. CONCLUSIONS: Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations

Metabarcoding data allow for reliable biomass estimates in the most abundant animals on earth

Schenk J, Geisen S, Kleinbölting N, Traunspurger W. Metabarcoding data allow for reliable biomass estimates in the most abundant animals on earth. Metabarcoding and Metagenomics. 2019;3: e46704.Microscopic organisms are the dominant and most diverse organisms on Earth. Nematodes, as part of this microscopic diversity, are by far the most abundant animals and their diversity is equally high. Molecular metabarcoding is often applied to study the diversity of microorganisms, but has yet to become the standard to determine nematode communities. As such, the information metabarcoding provides, such as in terms of species coverage, taxonomic resolution and especially if sequence reads can be linked to the abundance or biomass of nematodes in a sample, has yet to be determined. Here, we applied metabarcoding using three primer sets located within ribosomal rRNA gene regions to target assembled mock-communities consisting of 18 different nematode species that we established in 9 different compositions. We determined abundances and biomass of all species added to examine if relative sequence abundance or biomass can be linked to relative sequence reads. We found that nematode communities are not equally represented by the three different primer sets and we found that relative read abundances almost perfectly correlated positively with relative species biomass for two of the primer sets. This strong biomass-read number correlation suggests that metabarcoding reads can reveal biomass information even amongst more complex nematode communities as present in the environment and possibly can be transferred to better study other groups of organisms. This biomass-read link is of particular importance for more reliably assessing nutrient flow through food-webs, as well as adjusting biogeochemical models through user-friendly and easily obtainable metabarcoding data.</jats:p

An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana

Huep G, Kleinbölting N, Weisshaar B. An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods. 2014;10(1): 28.BACKGROUND: More than 90% of the Arabidopsis thaliana genes are members of multigene families. DNA sequence similarities present in such related genes can cause trouble, e.g. when molecularly analysing mutant alleles of these genes. Also, flanking-sequence-tag (FST) based predictions of T-DNA insertion positions are often located within paralogous regions of the genome. In such cases, the prediction of the correct insertion site must include careful sequence analyses on the one hand and a paralog specific primer design for experimental confirmation of the prediction on the other hand. RESULTS: GABI-Kat is a large A. thaliana insertion line resource, which uses in-house confirmation to provide highly reliable access to T-DNA insertion alleles. To offer trustworthy mutant alleles of paralogous loci, we considered multiple insertion site predictions for single FSTs and implemented this 1-to-N relation in our database. The resulting paralogous predictions were addressed experimentally and the correct insertion locus was identified in most cases, including cases in which there were multiple predictions with identical prediction scores. A newly developed primer design tool that takes paralogous regions into account was developed to streamline the confirmation process for paralogs. The tool is suitable for all parts of the genome and is freely available at the GABI-Kat website. Although the tool was initially designed for the analysis of T-DNA insertion mutants, it can be used for any experiment that requires locus-specific primers for the A. thaliana genome. It is easy to use and also able to design amplimers with two genome-specific primers as required for genotyping segregating families of insertion mutants when looking for homozygous offspring. CONCLUSIONS: The paralog-aware confirmation process significantly improved the reliability of the insertion site assignment when paralogous regions of the genome were affected. An automatic online primer design tool that incorporates experience from the in-house confirmation of T-DNA insertion lines has been made available. It provides easy access to primers for the analysis of T-DNA insertion alleles, but it is also beneficial for other applications as well

Enhancing the GABI-Kat Arabidopsis thaliana T-DNA insertion mutant database by incorporating Araport11 annotation

Kleinbölting N, Huep G, Weisshaar B. Enhancing the GABI-Kat Arabidopsis thaliana T-DNA insertion mutant database by incorporating Araport11 annotation. Plant and Cell Physiology. 2017;58(1): e7.SimpleSearch provides access to a database containing information about T-DNA insertion lines of the GABI-Kat collection of Arabidopsis thaliana mutants. These mutants are an important tool for reverse genetics, and GABI-Kat is the second largest collection of such T-DNA insertion mutants. Insertion sites were deduced from flanking sequence tags (FSTs), and the database contains information about mutant plant lines as well as insertion alleles. Here, we describe improvements within the interface (available at http://www.gabi-kat.de/db/genehits.php) and with regard to the database content that have been realized in the last five years. These improvements include the integration of the Araport11 genome sequence annotation data containing the recently updated A. thaliana structural gene descriptions, an updated visualization component that displays groups of insertions with very similar insertion positions, mapped confirmation sequences, and primers. The visualization component provides a quick way to identify insertions of interest, and access to improved data about the exact structure of confirmed insertion alleles. In addition, the database content has been extended by incorporating additional insertion alleles that were detected during the confirmation process, as well as by adding new FSTs that have been produced during continued efforts to complement gaps in FST availability. Finally, the current database content regarding predicted and confirmed insertion alleles as well as primer sequences has been made available as downloadable flat files

Dataset supporting the use of nematodes as bioindicators of polluted sediments

Schenk J, Höss S, Brinke M, Kleinbölting N, Brüchner-Hüttemann H, Traunspurger W. Dataset supporting the use of nematodes as bioindicators of polluted sediments. Data in Brief. 2020;32: 106087.We provide the dataset supporting the research article “Nematodes as bioindicators of polluted sediments using metabarcoding and microscopic taxonomy” [1]. Nematodes are frequently used as bioindicators and the NemaSPEAR[%] is an validated index that is originally based on morphological data. The index was compared to molecular sequence data for the 28S rDNA, 18S rDNA and COI gene for 7 locations. This dataset includes chemical analyses of the sediments for 33 different substances. The sequence data for OTU-based analyses for the 28S rDNA, 18S rDNA and COI gene is given, together with the read distribution during bioinformatics processing. We furthermore include alternative ASV data, based on a cluster-independent approach. The morphological data is presented, including the biomass for each species, as well as an overview about whether the species is represented in the NCBI database. Furthermore, rarefaction analysis is given for the morphological data, and furthermore NMDS plots for the species and genus level based on morphological and molecular data. The correlation between the mean PEC-Q and the NemaSPEAR[%] values is given in order to compare the efficiency of the index, based on morphological and molecular data

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

Pucker B, Kleinbölting N, Weisshaar B. Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genomics. 2021;22: 599.Background: Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results: We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions: Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations

Suitability of molecular taxonomy for assessing polluted sediments using the NemaSPEAR[%] index

Assessing the ecological consequences of marine and freshwater contamination is an important use of biological indicators. The NemaSPEAR[%] index is a nematode-based index for the evaluation of sediment quality and an expedient complement to macroinvertebrate-based indicator systems, especially for fine, cohesive sediments. While the NemaSPEAR[%] index in its original form is morphologically based, in this study the nematode communities of 38 locations with known sediment contamination were analyzed by light microscopy and metabarcoding to demonstrate the validity of a molecular-taxonomy-based NemaSPEAR[%] index. The results showed that the molecular-based index can reliably distinguish between sites of good, moderate, and bad pollution status. Moreover, a greater congruence with molecular read abundances were obtained after the morphology-based NemaSPEAR[%] index was corrected for differences in the biomass of different nematode species. However, incomplete reference databases hamper a full congruence between the species inventories recorded in morphological and molecular datasets. Another source of variability is the substantial difference in the biomass (and thus also of gene copies) of different species, thus evidencing a bias in abundance-based calculations of the NemaSPEAR[%] index. Despite these limitations, the molecular approach provides an expertise-free means of reliable bioindication using one of the most abundant and diverse components of benthic macroinvertebrate communities. Moreover, due to the ubiquity of nematodes, application of the index can be extended to wastewater, biofilm, and other lotic and lentic waters that require regular monitoring

Suitability of molecular taxonomy for assessing polluted sediments using the NemaSPEAR[%] index

Schenk J, Höss S, Kleinbölting N, Traunspurger W. Suitability of molecular taxonomy for assessing polluted sediments using the NemaSPEAR[%] index. Ecological Indicators. 2022;137: 108761