Kai, S. | Search bioRxiv

bioRxiv2024

Liquid footprinting: A novel approach for DNA footprinting using short double-stranded cell-free DNA from plasma

The diagnostic potential of short double-stranded cell-free DNA (cfDNA) in blood plasma has not been recognized, yet. Here, we present a method for enrichment of double-stranded cfDNA with an average length of about 40 base pairs from cfDNA for high-throughput DNA sequencing. This class of cfDNA is enriched at gene promoters and binding sites of transcription factors or structural DNA-binding proteins, so that a genome-wide DNA footprint is directly captured from liquid biopsies. In short double-stranded cfDNA from healthy individuals, we found significant enrichment of 203 transcription factor motifs. Additionally, short double-stranded cfDNA signals at specific genomic regions correlate negatively with DNA-methylation, positively with H3K4me3 histone modifications and gene transcription. When comparing short double-stranded cfDNA from patient samples of pancreatic ductal adenocarcinoma with colorectal carcinoma or septic with post-operative controls, we identified 731 and 1,107 differentially enriched loci, respectively. Using these differentially enriched loci, the disease types can be clearly distinguished by principal component analysis demonstrating the diagnostic potential of short double-stranded cfDNA signals as a new class of biomarkers for liquid biopsies.

genomics↗

bioRxiv2018

Direct PCR amplification of 16S rRNA genes offers accelerated bacterial identification using the MinION™ nanopore sequencer

Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable timeframe for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinIONTM sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

microbiology↗