Biology subjects

Kirkegaard, R.

Publications and source records attributed to Kirkegaard, R..

7 recordsLinked to original sources

bioRxiv2026

Sensitive long-read amplicon sequence variant recovery with savont

Long-read amplicon sequencing can profile longer sequences compared to short reads, but recovering amplicon sequence variants (ASVs) is a challenge for long, noisy reads. We present savont, an algorithm for recovering ASVs from modern long-read amplicons with mean accuracy [≥] 98%, including Oxford Nanopore Technologies (ONT) R10.4 and PacBio HiFi reads. Savont requires 5 to 16 times lower sequencing depth for full-length 16S rRNA ONT reads than previous methods and generates up to 95 times more ASVs for complex environments. Savont makes ASV-based analysis from shallow and noisy long-read amplicon sequencing feasible.

bioinformatics↗

bioRxiv2026

Nanopore sequencing reaches amplicon sequence variant (ASV) resolution

Sequencing of ribosomal marker genes remains a cornerstone for profiling complex microbial communities. In recent years, there has been a shift from Illumina to long-read technologies, including PacBio and Oxford Nanopore Technologies (ONT). ONT is attractive due to its low startup cost and portability; however, historically high error rates have prevented direct amplicon sequencing variant (ASV) generation from raw nanopore reads. This has forced most workflows to rely on mapping raw reads against reference databases constraining analyses to taxa covered by these. With recent improvements in ONT sequencing accuracy, we sought to challenge this view by sequencing samples of increasing complexity using primer sets targeting amplicons of different lengths, and by sequencing the exact same PCR libraries on both PacBio and ONT. We demonstrate that error-free ASVs can now be generated directly from raw nanopore reads using standard denoising algorithms originally developed for Illumina data. Current ONT read quality enables reliable reconstruction of amplicons spanning [~]250 bp to [~]4,200 bp and allows resolution of intragenomic rRNA gene variants. These results extend beyond simple mock communities to complex fecal, anaerobic digester, activated sludge, and soil samples. When sequencing depth is sufficient, ONT accurately recovers all or nearly all intra-genomic 16S rRNA gene copy variants, showing perfect sequence identity to curated reference sequences in mock communities and to ASVs inferred from PacBio data in complex communities. Across the primer sets, ONT required higher sequencing depth than PacBio to fully resolve the communities, with this requirement increasing with amplicon length. For complex samples, ONT required approximately 2-3x more reads for V4 ([~]250 bp) and V1-V3 ([~]500 bp), 4.1-5.6x more reads for V1-V8 ([~]1400 bp), and 25-42x more reads for rRNA operon (OPR) amplicons ([~]4200 bp). Consequently, sequencing complex communities with OPR primers on ONT is currently not feasible due to the unrealistically high read depth required. This study provides evidence that ONT amplicon sequencing has matured to the point where true ASV-resolved profiling is practically and economically feasible, moving ONT amplicon analysis beyond reliance on OTU clustering or reference alignment to enable application in environments lacking comprehensive reference databases. Key FindingsO_LIIt is now straightforward to generate ASVs on ONT platforms (250-4200 bp) C_LIO_LIONT can resolve intragenomic 16S rRNA gene variants C_LIO_LIASV recovery is successful in both simple and complex communities C_LI

bioinformatics↗

bioRxiv2026

A national baseline for methane sink habitats and methanotroph diversity

Methane emissions account for nearly a third of the Earths effective radiative forcing, with methanotrophs playing a critical role in mitigating emissions by oxidising methane in diverse environments1. Despite their ecological importance, methanotrophic diversity and environmental distribution remain incompletely characterised due to cultivation challenges, incomplete or low-quality metagenome-assembled genomes, and limited taxonomic resolution in marker gene surveys. Here, we present a national study of the biogeography of novel and known methanotrophs across Denmarks major natural, urban and agricultural habitats, using genome-resolved classification of 10,683 metagenomes2 and 102 new methanotrophic species3. By linking metabolic potential to habitat-specific distributions, we reveal uncharacterised methanotrophs as dominant in natural ecosystems. These findings provide a comprehensive baseline of methanotroph diversity, reveal clear contrasts between natural and disturbed habitats, and highlight candidate species and habitats for future methane-mitigation strategies.

microbiology↗

bioRxiv2024

Niche and spatial partitioning restrain ecological equivalence among microbes along aquatic redox gradient

Microbial metabolic capabilities and interactions shape their niche hypervolume that in turn governs their ecological strategies and ecosystem services. In the context of functional redundancy or ecological equivalence, the focus has been on functional guilds in order to bypass the complex challenge faced by niche theory for disentangling the niche hypervolume. However, in some cases this simplification has been at the expense of ignoring the role of individual genotype of each microbe within a functional guild and fails to explain how the diversity within each functional guild is maintained. In this study, we inspect the metabolic profile of metagenome-assembled genomes along the pronounced redox gradient of the water column in an anchialine cave. Bridging neutral theory of biodiversity and biogeography and niche theory, our analysis uses focal metabolic capabilities while also incorporating individuality by looking into background metabolic capabilities of each individual and further includes spatial distribution of microbes to delineate their niche space. Our results emphasize that differences in background metabolic capabilities are critical for furnishing the niche hypervolume of microbes carrying the same focal metabolic capability and refute their ecological equivalence with their spatial distribution further enables niche partitioning among them.

ecology↗

bioRxiv2024

Nanomotif: Identification and Exploitation of DNA Methylation Motifs in Metagenomes using Oxford Nanopore Sequencing

DNA methylation is found across all domains of life but is a rarely used feature in recovery of metagenome-assembled genomes (MAGs). Recently, Oxford Nanopore introduced all context methylation detection models. We leveraged this to develop Nanomotif, which identifies and exploits methylation motifs for enhanced MAG recovery. We demonstrate how Nanomotif enables database-independent contamination removal from high-quality MAGs and host association of plasmids directly from Nanopore sequencing data in complex metagenomes.

bioinformatics↗

bioRxiv2019

The signal and the noise - characteristics of antisense RNA in complex microbial communities

High-throughput sequencing has allowed unprecedented insight into the composition and function of complex microbial communities. With the onset of metatranscriptomics, it is now possible to interrogate the transcriptome of multiple organisms simultaneously to get an overview of the gene expression of the entire community. Studies have successfully used metatranscriptomics to identify and describe relationships between gene expression levels and community characteristics. However, metatranscriptomic datasets contain a rich suite of additional information which is just beginning to be explored. In this minireview we discuss the different computational strategies for handling antisense expression in metatranscriptomic samples and highlight their potentially detrimental effects on downstream analysis and interpretation. We also surveyed the antisense transcriptome of multiple genomes and metagenome-assembled genomes (MAGs) from five different datasets and found high variability in the level of antisense transcription for individual species which were consistent across samples. Importantly, we tested the hypothesis that antisense transcription is primarily the product of transcriptional noise and found mixed support, suggesting that the total observed antisense RNA in complex communities arises from a compounded effect of both random, biological and technical factors. Antisense transcription can provide a rich set of information, from technical details about data quality to novel insight into the biology of complex microbial communities.\n\nKey pointsO_LISeveral fundamentally different approaches are used to handle antisense RNA\nC_LIO_LIPrevalence of antisense RNA is highly variable between communities, genomes, and genes.\nC_LIO_LIAntisense RNA is likely an opaque mixture of technical, biological and random effects\nC_LI

microbiology↗

bioRxiv2019

Unlinked rRNA genes are widespread among Bacteria and Archaea

Ribosomes are essential to cellular life and the genes for their RNA components are the most conserved and transcribed genes in Bacteria and Archaea. These ribosomal rRNA genes are typically organized into a single operon, an arrangement that is thought to facilitate gene regulation. In reality, some Bacteria and Archaea do not share this canonical rRNA arrangement-their 16S and 23S rRNA genes are not co-located, but are instead separated across the genome and referred to as \"unlinked\". This rearrangement has previously been treated as a rare exception or a byproduct of genome degradation in obligate intracellular bacteria. Here, we leverage complete genome and long-read metagenomic data to show that unlinked 16S and 23S rRNA genes are much more common than previously thought. Unlinked rRNA genes occur in many phyla, most significantly within Deinococcus-Thermus, Chloroflexi, Planctomycetes, and Euryarchaeota, and occur in differential frequencies across natural environments. We found that up to 41% of the taxa in soil, including dominant taxa, had unlinked rRNA genes, in contrast to the human gut, where all sequenced rRNA genes were linked. The frequency of unlinked rRNA genes may reflect meaningful life history traits, as they tend to be associated with a mix of slow-growing free-living species and obligatory intracellular species. Unlinked rRNA genes are also associated with changes in RNA metabolism, notably the loss of RNaseIII. We propose that unlinked rRNA genes may confer selective advantages in some environments, though the specific nature of these advantages remains undetermined and worthy of further investigation.

microbiology↗