Biology subjects

Pucker, B.

Publications and source records attributed to Pucker, B..

68 records · Page 4Linked to original sources

bioRxiv2020

Reference-based QUantification Of gene Dispensability (QUOD)

BackgroundDispensability of genes in a phylogenetic lineage, e.g. a species, genus, or higher-level clade, is gaining relevance as most genome sequencing projects move to a pangenome level. Most analyses classify genes as core genes, which are present in all investigated individual genomes, and dispensable genes, which only occur in a single or a few investigated genomes. The binary classification as core or dispensable is often based on arbitrary cutoffs of presence/absence in the analysed genomes. Even when extended to conditionally dispensable, this concept still requires the assignment of genes to distinct groups. ResultsHere, we present a new method which overcomes this distinct classification by quantifying gene dispensability and present a dedicated tool for reference-based QUantification Of gene Dispensability (QUOD). As a proof of concept, sequence data of 966 Arabidopsis thaliana accessions (Ath-966) were processed to calculate a gene-specific dispensability score for each gene based on normalised coverage in read mappings. We validated this score by comparison of highly conserved Benchmarking Universal Single Copy Orthologs (BUSCOs) to all other genes. The average scores of BUSCOs were significantly lower than the scores of non-BUSCOs. Analysis of variation demonstrated lower variation values between replicates of a single accession than between iteratively, randomly selected accessions from the whole dataset Ath-966. Functional investigations revealed defense and antimicrobial response genes among the genes with high-dispensability scores. ConclusionsInstead of classifying a gene as core or dispensable, QUOD assigns a dispensability score to each gene. Hence, QUOD facilitates the identification of candidate dispensable genes, associated with high dispensability scores, which often underlie lineage-specific adaptation to varying environmental conditions.

bioinformatics↗

bioRxiv2020

Comparison of read mapping and variant calling tools for the analysis of plant NGS data

High-throughput sequencing technologies have rapidly developed during the past years and became an essential tool in plant sciences. However, the analysis of genomic data remains challenging and relies mostly on the performance of automatic pipelines. Frequently applied pipelines involve the alignment of sequence reads against a reference sequence and the identification of sequence variants. Since most benchmarking studies of bioinformatics tools for this purpose have been conducted on human datasets, there is a lack of benchmarking studies in plant sciences. In this study, we evaluated the performance of 50 different variant calling pipelines, including five read mappers and ten variant callers, on six real plant datasets of the model organism Arabidopsis thaliana. Sets of variants were evaluated based on various parameters including sensitivity and specificity. We found that all investigated tools are suitable for analysis of NGS data in plant research. When looking at different performance metrices, BWA-MEM and Novoalign were the best mappers and GATK returned the best results in the variant calling step.

bioinformatics↗

bioRxiv2020

The R2R3-MYB gene family in banana (Musa acuminata): genome-wide identification, classification and expression patterns

The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, defense responses and metabolite accumulation. To date MYB family genes have not yet been comprehensively identified in the major staple fruit crop banana. In this study, we present a comprehensive, genome-wide analysis of the MYB genes from Musa acuminata DH-Pahang (A genome). A total of 285 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Organ- and development-specific expression patterns were determined from RNA-seq data. For 280 M. acuminata MYB genes for which expression was found in at least one of the analysed samples, a variety of expression patterns were detected. The M. acuminata R2R3-MYB genes were functionally categorised, leading to the identification of seven clades containing only M. acuminata R2R3-MYBs. The encoded proteins may have specialised functions that were acquired or expanded in Musa during genome evolution. This functional classification and expression analysis of the MYB gene family in banana establishes a solid foundation for future comprehensive functional analysis of MaMYBs and can be utilized in banana improvement programmes.

plant biology↗

bioRxiv2020

High contiguity de novo genome sequence assembly of Trifoliate yam (Dioscorea dumetorum) using long read sequencing

Trifoliate yam (Dioscorea dumetorum) is one example of an orphan crop, not traded internationally. Post-harvest hardening of the tubers of this species starts within 24 hours after harvesting and renders the tubers inedible. Genomic resources are required for D. dumetorum to improve breeding for non-hardening varieties as well as for other traits. We sequenced the D. dumetorum genome and generated the corresponding annotation. The two haplophases of this highly heterozygous genome were separated to a large extent. The assembly represents 485 Mbp of the genome with an N50 of over 3.2 Mbp. A total of 35,269 protein-encoding gene models as well as 9,941 non-coding RNA genes were predicted and functional annotations were assigned.

genomics↗

bioRxiv2019

A Partially Phase-Separated Genome Sequence Assembly of the Vitis Rootstock 'Börner' (Vitis riparia x Vitis cinerea) and its Exploitation for Marker Development and Targeted Mapping

Grapevine breeding becomes highly relevant due to upcoming challenges like climate change, a decrease in the number of available fungicides, increasing public concern about plant protection, and the demand for a sustainable production. Downy mildew caused by Plasmopara viticola is one of the most devastating diseases worldwide of cultivated Vitis vinifera. Therefore, in modern breeding programs genetic marker technologies and genomic data are used to develop new cultivars with defined and stacked resistance loci. Potential sources of resistance are wild species of American or Asian origin. The interspecific hybrid of Vitis riparia Gm 183 x V. cinerea Arnold, available as the rootstock cultivar Borner, carries several relevant resistance loci. We applied next generation sequencing to enable the reliable identification of simple sequence repeats (SSR) and also generated a draft genome sequence assembly of Borner to access genome wide sequence variations in a comprehensive and highly reliable way. These data were used to cover the Borner genome with genetic marker positions. A subset of these marker positions was used for targeted mapping of the P. viticola resistance locus, Rpv14, to validate the marker position list. Based on the reference genome sequence PN40024, the position of this resistance locus can be narrowed down to less than 0.5 Mbp on chromosome 5.

genomics↗

bioRxiv2019

25 years of propagation in suspension cell culture results in substantial alterations of the Arabidopsis thaliana genome

Arabidopsis thaliana is one of the best studied plant model organisms. Besides cultivation in greenhouses, cells of this plant can also be propagated in suspension cell culture. At7 is one such cell line that has been established about 25 years ago. Here we report the sequencing and the analysis of the At7 genome. Large scale duplications and deletions compared to the Col-0 reference sequence were detected. The number of deletions exceeds the number of insertions thus indicating that a haploid genome size reduction is ongoing. Patterns of small sequence variants differ from the ones observed between A. thaliana accessions e.g. the number of single nucleotide variants matches the number of insertions/deletions. RNA-Seq analysis reveals that disrupted alleles are less frequent in the transcriptome than the native ones.

genomics↗

bioRxiv2019

Genome sequencing of Musa acuminata Dwarf Cavendish reveals a chromosome arm duplication

Different Musa species, subspecies, and cultivars are currently investigated to reveal their genomic diversity. Here, we compare the genome sequence of one of the commercially most important cultivars, Musa acuminata Dwarf Cavendish, against the Pahang reference genome assembly. Numerous small sequence variants were detected and the ploidy of the cultivar presented here was determined as triploid based on sequence variant frequencies. Illumina sequence data also revealed a duplication of a large segment on the long arm of chromosome 2 in the Dwarf Cavendish genome. Comparison against previously sequenced cultivars provided evidence that this duplication is unique to Dwarf Cavendish. Although no functional relevance of this duplication was identified, this example shows the potential of plants to tolerate such aneuploidies.

genomics↗

bioRxiv2019

Next generation sequencing to investigate genomic diversity in Caryophyllales

Caryophyllales are a highly diverse and large order of plants with a global distribution. While some species are important crops like Beta vulgaris, many others can survive under extreme conditions. This order is well known for the complex pigment evolution, because the pigments anthocyanins and betalains occur with mutual exclusion in species of the Caryophyllales. Here we report about genome assemblies of Kewa caespitosa (Kewaceae), Macarthuria australis (Macarthuriaceae), and Pharnaceum exiguum (Molluginaceae) which are representing different taxonomic groups in the Caryophyllales. The availability of these assemblies enhances molecular investigation of these species e.g. with respect to certain genes of interest.

genomics↗

bioRxiv2019

Animal, fungi, and plant genome sequences harbour different non-canonical splice sites

Most protein encoding genes in eukaryotes contain introns which are interwoven with exons. After transcription, introns need to be removed in order to generate the final mRNA which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5 end and AG at the 3 end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations which have been known for years. During the last years, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here we expand systematic investigations of non-canonical splice site combinations in plants to all eukaryotes by analysing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences such as a substantially increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts could be one explanation for this observation. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 snRNA isoform might allow the recognition of GA as 5 splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3 splice site compared to the 5 splice site across animals, fungi, and plants.

genomics↗

bioRxiv2019

Mapping-based genome size estimation

While the size of chromosomes can be measured under a microscope, obtaining the exact size of a genome remains a challenge. Biochemical methods and k-mer distribution-based approaches allow only estimations. An alternative approach to estimate the genome size based on high contiguity assemblies and read mappings is presented here. Analyses of Arabidopsis thaliana and Beta vulgaris data sets are presented to show the impact of different parameters. Oryza sativa, Brachypodium distachyon, Solanum lycopersicum, Vitis vinifera, and Zea mays were also analyzed to demonstrate the broad applicability of this approach. Further, MGSE was also used to analyze Eschericia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans datasets to show its utility beyond plants. Mapping-based Genome Size Estimation (MGSE) and additional scripts are available on GitHub: https://github.com/bpucker/MGSE. MGSE predicts genome sizes based on short reads or long reads requiring a minimal coverage of 5-fold.

genomics↗

bioRxiv2019

Influence of neighboring small sequence variants on functional impact prediction

Once a suitable reference sequence has been generated, intraspecific variation is often assessed by re-sequencing. Variant calling processes can reveal all differences between strains, accessions, genotypes, or individuals. These variants can be enriched with predictions about their functional implications based on available structural annotations, i.e. gene models. Although these functional impact predictions on a per-variant basis are often accurate, some challenging cases require the simultaneous incorporation of multiple adjacent variants into this prediction process. Examples include neighboring variants which modify each others functional impact. The Neighborhood-Aware Variant Impact Predictor (NAVIP) considers all variants within a given protein coding sequence when predicting the effect. As a proof of concept, variants between the Arabidopsis thaliana accessions Columbia-0 and Niederzenz-1 were annotated. NAVIP is freely available on GitHub (https://github.com/bpucker/NAVIP) and accessible through a web server (https://pbb-tools.de). Author SummaryIntraspecific variation gains increasing relevance as reference genome sequences are available for many investigated (plant) species. Understanding the effects of sequence variants between individuals of a population is a challenge. SnpEff (Cingolani et al., 2012) is the current standard tool for predicting the functional impact of sequence variants, but only considers one sequence variant at a time. We developed NAVIP to properly handle cases in which multiple sequence variants cluster together and influence each others functional impact. A comparison of two Arabidopsis thaliana accessions demonstrates the importance of considering multiple sequence variants simultaneously for the prediction of changes in encoded proteins. NAVIP is universally applicable to any organism for which the relevant sequence information and structural annotation is available. All underlying code is freely available on GitHub and we operate a web server for users convenience.

genomics↗

bioRxiv2018

Genome-wide analyses supported by RNA-Seq reveal non-canonical splice sites in plant genomes

Most eukaryotic genes comprise exons and introns thus requiring the precise removal of introns from pre-mRNAs to enable protein biosynthesis. U2 and U12 spliceosomes catalyze this step by recognizing motifs on the transcript in order to remove the introns. A process which is dependent on precise definition of exon-intron borders by splice sites, which are consequently highly conserved across species. Only very few combinations of terminal dinucleotides are frequently observed at intron ends, dominated by the canonical GT-AG splice sites on the DNA level.\n\nHere we investigate the occurrence of diverse combinations of dinucleotides at predicted splice sites. Analyzing 121 plant genome sequences based on their annotation revealed strong splice site conservation across species, annotation errors, and true biological divergence from canonical splice sites. The frequency of non-canonical splice sites clearly correlates with their divergence from canonical ones indicating either an accumulation of probably neutral mutations, or evolution towards canonical splice sites. Strong conservation across multiple species and non-random accumulation of substitutions in splice sites indicate a functional relevance of non-canonical splice sites. The average composition of splice sites across all investigated species is 98.7% for GT-AG, 1.2% for GC-AG, 0.06% for AT-AC, and 0.09% for minor non-canonical splice sites. RNA-Seq data sets of 35 species were incorporated to validate non-canonical splice site predictions through gaps in sequencing reads alignments and to demonstrate the expression of affected genes. We conclude that bona fide non-canonical splice sites are present and appear to be functionally relevant in most plant genomes, if at low abundance.

genomics↗

bioRxiv2018

Harnessing natural diversity to identify key residues in Prolidase

Prolidase (PEPD) catalyses the cleavage of dipeptides with high affinity for proline at the C-terminal end. This function is required in almost all living organisms. In order to detect strongly conserved residues in PEPD, we analysed PEPD orthologous sequences identified in data sets of animals, plants, fungi, archaea, and bacteria. Due to conservation over very long evolutionary time, conserved residues are likely to be of functional relevance. Single amino acid mutations in PEPD cause a disorder called prolidase deficiency and are associated with various cancer types. We provide new insights into 15 additional residues with putative roles in prolidase deficiency and cancer. Moreover, our results confirm previous reports identifying five residues involved in the binding of metal cofactors as highly conserved and enable the classification of several non-synonymous single nucleotide polymorphisms as likely pathogenic and seven as putative polymorphisms. Moreover, more than 50 novel conserved residues across species were identified. Conservation degree per residue across the animal kingdom were mapped to the human PEPD 3D structure revealing the strongest conservation close to the active site accompanied with a higher functional implication and pathogenic potential, validating the importance of a characteristic active site fold for prolidase identity.

evolutionary biology↗

bioRxiv2018

A Chromosome-level Sequence Assembly Reveals the Structure of the Arabidopsis thaliana Nd-1 Genome and its Gene Set

BackgroundIn addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species.\n\nResultsHere we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences.\n\nConclusionsDetailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome.

genomics↗