Biology subjects

Spohr, P.

Publications and source records attributed to Spohr, P..

5 recordsLinked to original sources

bioRxiv2026

Barcode Crosstalk in ONT Multiplex Sequencing: Quantification and Mitigation Strategies

Multiplexing samples in long-read sequencing with Oxford Nanopore Next Generation Sequencing Technology (ONT) by ligating specific native barcodes to individual DNA samples enables significant increases of high throughput sequencing combined with a significant reduction of sequencing costs. However, this advantage carries the risk of barcode misassignment / crosstalk. Employing ONT multiplex sequencing with samples, we observed misassigned barcodes so called barcode crosstalk, after ONT library preparation according to the standard protocol, particularly in samples with low input DNA concentrations. We assumed that these barcode misassignments are largely due to misligation of remaining native barcodes during subsequent the subsequent sequencing adapter ligation. To systematically investigate and quantify barcode crosstalk, genomic DNA (gDNA) from four bacterial type strains with different DNA input concentrations was prepared using three protocols for library preparation: the Nanopore standard protocol (protocol A: version valid until July 2, 2025) the new Nanopore protocol (protocol B: version from July 2, 2025), and an in house protocol with pooling of the barcoded samples only after the sequencing adapter ligation step (protocol C: in house). All samples were sequenced on a Nanopore PromethIon device. The results clearly showed that the use of protocol A resulted in a pronounced barcode crosstalk especially detectable in samples with low DNA input concentrations (up to 2.4% misassigned reads). The ONT adjustment in protocol B (altered washing buffer vs. protocol A) significantly alleviated the barcode crosstalk to below 0.01%, whereas protocol C eliminated barcode crosstalk virtually completely. These observations emphasize that sequencing results obtained with older ONT native barcoding protocol variants should be critically reviewed. The newer ONT barcoding protocol is preferable for sequencing, but it does not completely eliminate the barcode crosstalk effect. In conclusion, for low DNA input and high accuracy sequencing, protocol C is recommended.

molecular biology↗

bioRxiv2023

CATHI: An interactive platform for comparative genomics and homolog identification

AO_SCPLOWBSTRACTC_SCPLOWBioinformatics has established itself as a central pillar of modern biology. Specifically, comparative genomics enables scientists to study a vast number of genomes efficiently. These comparative analyses shed light on the evolution and potential function of genomes and genes, but are also increasingly used as a key tool for metabolic engineering and synthetic biology by identifying appropriate targets for modification. While numerous sophisticated tools for comparative genomics and homolog identification exist, those tools predominantly target highly skilled bioinformatics users. Consequently, many biologists either defer such analyses to their more versed bioinformatic collaborators or resort to suboptimal tools. Here, we present an intuitive solution available on all major operating systems, easily accessed through common web browsers. CATHI - Comparative Analysis Tool for Homolog Identification - integrates a suite of best-practice bioinformatic tools, encompassing BLAST for homology searches, MAFFT for multiple sequence alignment, FastTree2 for phylogeny reconstruction, and clinker for synteny analysis. Specifically tailored to biologists, CATHI orchestrates predefined settings and automated pipelines, obviating the need for programming expertise. This platform empowers researchers to confidently engage in detailed comparative genomics studies by streamlining the analytical process. The interactive framework provides users with a plethora of options. This includes real-time execution and progress monitoring, facilitates dynamic result tracking, and a set of search functions across NCBI databases like CDD or ProtFam. Users can interactively engage in data exploration, filtering, and visualization through CATHIs intuitive interface. Furthermore, the seamless export of project data in standard formats (FASTA, Newick, CSV, and HTML) facilitates the integration with further third-party tools such as TreeViewer and Jalview. To benchmark CATHI, we revisited the comparative analysis of cyanobacterial circadian clock proteins conducted by Schmelling et al. in 2017, revealing consistent global patterns among identified homologs, while also highlighting individual variations attributed to the expansion of available databases.

bioinformatics↗

bioRxiv2023

MetaGut: Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Up to recently, exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members was limited due to 16S rDNA sequencing. Here, we developed MetaGut, a method enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using MetaGut we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, associated with Bacteroides/Phocaeicola, mixed composition and Enterococcus abundances. MetaGut revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples (up to >50% and >20%, respectively). After leukopenia, strains were stable or newly acquired. Our results demonstrate the disruptive effect of alloHSCT on the gut microbiome and pave the way for future studies based on long-read metagenomics.

bioinformatics↗

bioRxiv2021

Rhizobiales commensal bacteria promote Arabidopsis thaliana root growth via host sulfated peptide pathway

Root-associated commensal bacteria that belong to the order Rhizobiales, which also contains symbiotic and pathogenic bacteria, promote primary root growth of Arabidopsis thaliana. However, the molecular mechanism underlying this root growth promotion (RGP) activity remained unclear. Here, we conducted a transcriptomic analysis of A. thaliana roots inoculated with root-associated commensal bacteria of Rhizobiales and sister lineages and revealed common and strain/lineage-specific transcriptional response, possibly mediated by WRKY and ANAC family of transcription factors. We showed that the observed common response was also partly triggered by a wide range of non-pathogenic bacteria, fungi, and a multikingdom synthetic community (SynCom). This response was characterized by a down-regulation of genes related to intracellular redox regulation, suggesting distinctive redox status between pathogenic and non-pathogenic interactions. By integrating with developmental and cell biological experiments, we identified a crucial role of TYROSYLPROTEIN SULFOTRANSFERASE (TPST) in Rhizobiales RGP. Conversely, none of the known TPST-dependent sulfated peptide pathways appeared to be required for this activity, suggesting an unidentified component in the protein sulfation pathway targeted by Rhizobiales RGP. Finally, we show that TPST is needed for RGP exerted by Rhizobiales but not Pseudomonadales isolates, delineating lineage-specific mechanisms to manipulate host root development.

plant biology↗

bioRxiv2020

The Homo-Edit Distance Problem

We consider the homo-edit distance problem, which is the minimum number of homo-deletions or homo-insertions to convert one string into another. A homo-insertion is the insertion of a string of equal characters into another string, while a homo-deletion is the inverse operation. We show how to compute the homo-edit distance of two strings in polynomial time: We first demonstrate that the problem is equivalent to computing a common subsequence of the two input strings with a minimum number of homo-deletions and then present a dynamic programming solution for the reformulated problem. 2012 ACM Subject ClassificationApplied computing [->] Bioinformatics; Applied computing [->] Molecular sequence analysis; Theory of computation [->] Dynamic programming

bioinformatics↗