Biology subjects

Heeger, F.

Publications and source records attributed to Heeger, F..

4 recordsLinked to original sources

bioRxiv2020

Identification of enzymes for lignocellulose degradation in the genome and transcriptome of the aquatic hyphomycete Clavariopsis aquatica.

Fungi are ecologically important decomposers of lignocellulose. Basidiomycetes use peroxidases, laccases, and enzymes of the cytochrome P450 superfamily for cometabolic lignin degradation in order to access cellulose and hemicellulose as carbon sources. Limited lignin modification capabilities have also been reported for some terrestrial ascomycetes. Here we newly sequenced the genome of an exclusively aquatic ascomycete, Clavariopsis aquatica, documented the presence of genes for the modification of lignocellulose and its constituents, and compared differential gene expression between C. aquatica cultivated on lignocellulosic and sugar-rich substrates. We identified potential peroxidases, laccases, and cytochrome P450 monooxygenases several of which were differentially expressed when experimentally grown on different substrates. Additionally, we found regulation of pathways for cellulose and hemicellulose degradation. Our results suggest that C. aquatica is able to modify lignin, detoxify aromatic lignin constituents, or both. This may facilitate the use of carbohydrate components of lignocellulose as carbon and energy sources.

ecology↗

bioRxiv2020

Optimization of ddRAD-like data leads to high quality sets of reduced representation single copy orthologs (R2SCOs) in a sea turtle multi-species analysis.

Reduced representation libraries (RRS) allow large scale studies on non-model species to be performed without the need for a reference genome, by building a pseudo-reference locus catalog directly from the data. However, using closely-related high-quality genomes can help maximize nucleotide variation identified from RRS libraries. While chromosome-level genomes remain unavailable for most species, researchers can still invest in building high-quality and project-specific de novo locus catalogs. Among methods that use restriction enzymes (RADSeq), those including fragment size selection to help obtain the desired number of loci - such as double-digest RAD (ddRAD) - are highly flexible but can present important technical issues. Inconsistent size selection reproducibility across libraries and variable coverage across fragment lengths can affect genotyping confidence, number of identified single nucleotide polymorphisms (SNPs), and quality and completeness of the de novo reference catalog. We have developed a strategy to optimize locus catalog building from ddRAD-like data by sequencing overlapping reads that recreate original fragments and add information about coverage per fragment size. Further in silico size selection and digestion steps limit the filtered dataset to well-covered sets of loci and identity thresholds are estimated based on sequence pairwise comparisons. We have developed a full workflow that identifies a set of reduced-representation single-copy orthologs (R2SCOs) for any given species and that includes estimating and evaluating allelic variation in comparison with SNP calling results. We also show how to use our concept in an established RADSeq pipeline - Stacks - and confirm that our approach increases average coverage and number of SNPs called per locus in the final catalog. We have demonstrated our full workflow using newly generated data from five sea turtle species and provided further proof-of-principle using published hybrid sea turtle and primate datasets. Finally, we showed that a project-specific set of R2SCOs perform better than a draft genome as a reference.

evolutionary biology↗

bioRxiv2019

Combining the 5.8S and ITS2 gene regions to improve classification of fungi

O_LIThe internal transcribed spacer (ITS) is used in DNA metabarcoding of fungi. One disadvantage of its high variability may be a failure to classify OTUs when no similar reference sequence exists. We tested whether the 5.8S region, often sequenced with ITS2 but discarded before analysis, could provide OUT classifications when ITS fails. C_LIO_LIWe used in silico evaluation to compare classification success of 5.8S and ITS from the UNITE database when reference sequences of the same species, genus, or family were removed. We then developed an automated pipeline for a combined 5.8S - ITS2 analysis and applied it to mixed environmental samples containing many lineages that are underrepresented in databases. C_LIO_LIITS was clearly superior for species-level classifications with a complete reference database, but 5.8S outperformed ITS at higher level classifications with an incomplete database. Our combined 5.8S-ITS2 pipeline classified 3x more fungal OTUs compared to ITS2 alone, particularly within Chytridiomycota (10x) and Rozellamycota (3x). C_LIO_LIMissing reference sequences led to the failure of ITS to classify many fungal OTUs at all, and to a significant underestimation of environmental fungal diversity. Using 5.8S to complement ITS classification will likely provide better estimates of diversity in lineages for which database coverage is poor. C_LI

ecology↗

bioRxiv2018

Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

DNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well-established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels 15 comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By 20 allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 25 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.

ecology↗