Biology subjects

Kucklick, M.

Publications and source records attributed to Kucklick, M..

5 recordsLinked to original sources

bioRxiv2026

A framework for peptide identification on commercial nanopore sequencing platforms

Direct single-molecule peptide analysis could in principle enable rapid and sensitive identification of pathogen-derived or disease-associated biomarkers without reliance on mass spectrometry. However, existing nanopore peptide sensing methods are typically constrained by limited throughput and lack of accessibility beyond specialized setups. Here, we present an integrated experimental-computational framework for DNA-linked peptide translocation on a commercially available, high-throughput nanopore sequencing platform, the MinION. Synthetic peptides were covalently bound to oligonucleotides at both termini. The resulting peptide-DNA constructs were then translocated through the CsgG-CsgF pores using a DNA motor protein. Current traces were segmented using the known DNA sequences to extract peptide-associated signal regions. From these segments, we extracted signal features and trained feature-based and deep-learning classifiers to distinguish peptides, balancing interpretability and classification performance. We establish a framework for peptide identification using standard nanopore sequencing hardware. Across a diverse panel of synthetic peptides, our approach resolves single-amino-acid substitutions, maintains performance across independent sequencing runs, and correctly identifies peptides in blind mixtures. Interpretable model analyses connect classifier decisions and common errors to specific signal motifs. By combining commercially available instrumentation with a reproducible experimental and computational workflow, this framework lowers the barrier to nanopore-based proteomics and enables broader adoption across laboratories. It provides a foundation for future developments in amino acid modification detection and sequence analysis.

bioinformatics↗

bioRxiv2024

End-to-end simulation of nanopore sequencing signals with feed-forward transformers

MotivationNanopore sequencing represents a significant advancement in genomics, enabling direct long-read DNA sequencing at the single-molecule level. Accurate simulation of nanopore sequencing signals from nucleotide sequences is crucial for method development and for complementing experimental data. Most existing approaches rely on predefined statistical models, which may not adequately capture the properties of experimental signal data. Furthermore, these simulators were developed for earlier versions of nanopore chemistry, which limits their applicability and adaptability to the latest flow cell data. ResultsTo enhance the quality of artificial signals, we introduce seq2squiggle, a novel transformer-based, non-autoregressive model designed to generate nanopore sequencing signals from nucleotide sequences. Unlike existing simulators that rely on static k-mer models, our approach learns sequential contextual information from the signal data. We benchmark seq2squiggle against state-of-the-art simulators on real experimental R10.4.1 data, evaluating signal similarity, basecalling accuracy, and variant detection rates. Seq2squiggle consistently outperforms existing tools across multiple datasets, demonstrating superior similarity to real data and offering a robust solution for simulating nanopore sequencing signals with the latest flow cell generation. Availability and Implementationseq2squiggle is freely available on GitHub at: github.com/ZKI-PH-ImageAnalysis/seq2squiggle

bioinformatics↗

bioRxiv2022

Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogen Campylobacter jejuni

BackgroundKnowing the molecules encoded by bacterial pathogens and how their expression is regulated is essential to understand how they survive, colonize, and cause disease. RNA-seq technologies that map transcriptomes have revealed a wealth of new transcripts in bacterial pathogens. However, they do not provide direct evidence or coordinates for coding potential. In particular, they miss small proteins ([≤] 50-100 amino acids) translated from small open reading frames (sORFs). However, this still poorly annotated component of bacterial genomes shows emerging roles in bacterial physiology and virulence. ResultsHere, we present an integrated approach based on complementary ribosome profiling (Ribo-seq) techniques to map the "translatome" of Campylobacter jejuni, the most common cause of bacterial gastroenteritis. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and reveal internal sORFs. We also developed a Ribo-seq approach for mapping of translation termination sites (TTS), which revealed stop codons not apparent from the reference genome in virulence-associated loci. Our translatome map confirms translation of leaderless ORFs and leader peptides, re-annotates start or stop codons of 35 genes, and reveals isoforms generated by internal start sites. It also adds 42 novel sORFs in diverse contexts to the C. jejuni annotation, such as within small RNAs, in 5' untranslated regions (UTRs), or internal/out-of-frame/antisense in larger ORFs. Using epitope tagging/western blot and mass spectrometry we validated expression of almost 60 annotated and novel sORFs, including cioY, which we show encodes a conserved, 34 amino acid component of the CioAB terminal oxidase. ConclusionsOverall, we provide a blueprint for integrating several Ribo-seq approaches to refine and enrich bacterial annotations.

genomics↗

bioRxiv2020

A proteogenomics workflow to uncover the world of small proteins in Staphylococcus aureus

Small proteins play diverse and essential roles in bacterial physiology and virulence. Despite their importance, automated genome annotation algorithms still cannot accurately annotate all respective small open reading frames (sORFs), as they usually provide insufficient sequence information for domain and homology searches, tend to be species specific and only a few experimentally validated examples are covered in standard proteomics studies. The accuracy and reliability of genome annotations, particularly for sORFs, can be significantly improved by integrating protein evidence from experimental approaches that enrich for small proteins. Here we present a highly optimized and flexible workflow for bacterial proteogenomics, which covers all steps from (i) creation of protein databases, (ii) database searches, (iii) peptide-to-genome mapping to (iv) result interpretation and whose automated execution is supported by two open source tools (SALT & Pepper). We used the workflow to identify high quality peptide spectrum matches (PSMs) for both annotated and unannotated small proteins ([≤] 100 aa; SP100) in Staphylococcus aureus Newman. Proteins isolated from cells at the exponential and stationary growth phase were digested with different endopeptidases (trypsin, Lys-C, AspN), the resulting peptides fractionated by gel-based and gel-free methods and measured with highly sensitive mass spectrometers. PSMs or sORF predictions from sORFfinder were stringently filtered allowing us to detect 185 soluble SP100, 69 of which were missing in the used genome annotation. Most interestingly, almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids. In addition, phage-related functions were proposed for 30 SP100, based on the localization of their coding sequences in the genome.

genomics↗

bioRxiv2019

Dinoroseobacter shibae outer membrane vesicles are enriched for the chromosome dimer resolution site dif

Outer membrane vesicles (OMVs) of Gram-negative bacteria have key roles in pathogenesis. However, little is known about their biogenesis and cargo in marine bacteria. In Dinoroseobacter shibae, a marine member of the Rhodobacteraceae, OMVs were produced throughout exponential growth, and DNA could be detected by fluorescence microscopy inside appr. 65% of vesicles. Single cell analysis using time-lapse microscopy showed that individual cells secreted multiple OMVs, preferentially at the septum during cell division. OMVs were enriched for saturated fatty acids, thus their secretion likely increases the fluidity of the membrane of the releasing cell locally. DNA was isolated from the vesicle lumen and sequenced; it was up to 40fold enriched for the region around the terminus of replication (ter). Within this region, the peak of coverage of vesicle DNA was located at dif, a conserved 28 bp palindromic sequence required for binding of the site specific tyrosine recombinases XerCD which are activated by the divisome protein FtsK immediately prior to septum formation. Some of the most abundant proteins of the vesicle proteome were predicted to be required for direct interaction with peptidoglycan during cell division. Single cell analysis, electron microscopy, proteome and DNA cargo show that constitutive OMV secretion in D. shibae occurs mainly prior to septum formation. The footprint of the FtsK/XerCD molecular machinery which resolves chromosome dimers suggests a novel highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of this type of vesicles.

microbiology↗