Metagenomics analysis for microbial ecology investigation on historical samples: negligible effect of host DNA and optimal analysis strategies

Abstract

9 10 Microbiome composition and function are shaped by its environment with major shifts 11 induced by the intensified anthropogenic pressures over the past centuries. This time interval 12 extends beyond experimental and longitudinal studies typically designed to investigate 13 microbiome change. The extensive collection of historical samples available in museums and 14 herbaria worldwide is an untapped resource for studying host-microbiome interactions over a 15 prolonged time and in a global scale. Currently, their potential remains unfulfilled owing to 16 incompatibilities with analytical pipelines and limited understanding of optimal classification 17 parameters. Host DNA removal was deemed necessary for accurate taxonomic assignment of 18 metagenomic reads, however, for many host species in historical samples it is impractical due to 19 the absence of reference genomes. Here, we demonstrated that host DNA content has no significant 20 impact on key microbial ecological metrics such as alpha- and beta-diversity. In addition, 21 sequencing reads from historical samples are often highly fragmented due to degradation 22 processes. Through k-mer analysis of genomics sequences from host species and their associated 23 microbiomes, we observed that the shortest length of metagenomics read that can produce 24 meaningful results for taxonomic profiling in historical samples is 21 bp. Overall, this study 25 establishes a solid foundation for integrating extensive natural history collections into host- 26 associated microbiome research, offering important insights into the anthropogenic impacts on 27 microbiomes. 28 bioRxiv preprint doi: https://doi.org/10.1101/2025.04.05.647358; this version posted April 6, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. 29