Abstract Title
From trace to treasure. Optimizing RNA extraction for Avian Metagenomics
Abstract
The advancement of high-throughput sequencing technologies has enabled the study of the bacteriome and virome in wild birds. Specifically, metatranscriptomics is a key method for virome analysis and could be very useful in determining coinfections in avian influenza-positive animals. RNA sequencing demands careful sample handling and processing due to the lack of stability of RNA in the environment. However, these conditions are sometimes challenging to achieve. The main objective of this study was to evaluate the efficiency of three commercial RNA extraction kits for biological samples obtained from 6 wild bird species (Black-headed Gull, Griffon vulture, House sparrow, Mallard, Song Thrush, Spotless Starling). We extracted 24 samples (faeces, oral swabs, and cloacal swabs) using RNeasy Power Microbiome, RNeasy Power Fecal Pro, and RNeasy Mini Kit (Qiagen). The extraction performance was assessed based on total RNA yield, RNA integrity, and purity ratios (A260/280; A260/230). Overall, the RNeasy Power Fecal Pro kit performed best across most samples and species, except for Song Thrush, for which better performance was obtained with the RNeasy Mini Kit. Faeces were the optimal sample type for RNA extraction, while oral swabs yielded the least RNA. These findings underscore the significance of selecting optimised RNA extraction methods when studying wild avian viromes, as kit performance can vary significantly by species and sample type. These insights not only enhance methodological rigour in viromics, but also provide a foundation for future research on virus ecology and host-pathogen interactions in wildlife.
Funding: MCIN/AEI/10.13039/501100011033, FSE+ (PID2020-114060RR-C31 and PRE2022-101762); Basque Government.
Funding: MCIN/AEI/10.13039/501100011033, FSE+ (PID2020-114060RR-C31 and PRE2022-101762); Basque Government.
Co-Author(s)
Ane López-Morales, Vega Alvarez, Xeider Gerrikagoitia, Jose Luis Lavín and Marta Barral
Animal Health Department. NEIKER – Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA). 48160 Derio (Bizkaia). SPAIN
Abstract Category
Mortality estimations, impacts on harvest, conservation considerations, and potential mitigation strategies in wild birds