Drosou, K. | Search bioRxiv

bioRxiv2023

A novel integrated extraction protocol for multi-omic studies in heavily degraded samples

The combination of multi-omic techniques, e.g. genomics, transcriptomics, proteomics, metabolomics and epigenomics has revolutionised studies in medical research. These are employed to support biomarker discovery, better understand molecular pathways and identify novel drug targets. Despite concerted efforts in integrating omic datasets, there is an absence for the integration of all four biomolecules in a single extraction protocol. Here, we demonstrate for the first time a novel, minimally destructive integrated protocol for the simultaneous extraction of artificially degraded DNA, proteins, lipids and metabolites from pig brain samples. We used an MTBE-based approach to separate lipids and metabolites, followed by subsequent isolation of DNA and proteins. We have validated this protocol against standalone extraction protocols and show comparable or higher yield of all four biomolecules. This integrated protocol is key towards facilitating preservation of irreplaceable samples while promoting downstream analyses and successful data integration by removing bias from univariate dataset noise and varied distribution characteristics.

evolutionary biology↗

bioRxiv2020

An open-sourced bioinformatic pipeline for the processing of Next-Generation Sequencing derived nucleotide reads: Identification and authentication of ancient metagenomic DNA.

Bioinformatic pipelines optimised for the processing and assessment of metagenomic ancient DNA (aDNA) are needed for studies that do not make use of high yielding DNA capture techniques. These bioinformatic pipelines are traditionally optimised for broad aDNA purposes, are contingent on selection biases and are associated with high costs. Here we present a bioinformatic pipeline optimised for the identification and assessment of ancient metagenomic DNA without the use of expensive DNA capture techniques. Our pipeline actively conserves aDNA reads, allowing the application of a bioinformatic approach by identifying the shortest reads possible for analysis (22-28bp). The time required for processing is drastically reduced through the use of a 10% segmented non-redundant sequence file (229 hours to 53). Processing speed is improved through the optimisation of BLAST parameters (53 hours to 48). Additionally, the use of multi-alignment authentication in the identification of taxa increases overall confidence of metagenomic results. DNA yields are further increased through the use of an optimal MAPQ setting (MAPQ 25) and the optimisation of the duplicate removal process using multiple sequence identifiers (a 4.35-6.88% better retention). Moreover, characteristic aDNA damage patterns are used to bioinformatically assess ancient vs. modern DNA origin throughout pipeline development. Of additional value, this pipeline uses open-source technologies, which increases its accessibility to the scientific community.

genomics↗