DNA Extraction Protocols for Animal Fecal Material on Blood Spot Cards

Abstract

BackgroundCollecting fecal samples using dry preservatives is an attractive option in large epidemiological studies as they are easy to use, cheap and independent of cold chain logistics. Here, we test four DNA extraction methods with the aim of identifying an efficient procedure to extract high-quality DNA from fecal material of canine, sheep, equine, bovine, and pig collected on dry blood spot cards, with the goal of generating good quality shotgun metagenomics datasets. Further, the suitability of Illumina shotgun metagenomic sequencing at 20million PE read depth was assessed on its ability to successfully characterize the taxonomic and functional aspects of the resulting fecal microbiome. MethodsDNA was extracted from pig feces and mock communities collected on blood spot cards using four DNA extraction methods; two different methods of the QIAsymphony(R) PowerFecal(R) Pro DNA Kit, the ZymoBIOMICS DNA Miniprep Kit, and the MagNA Pure 96 DNA and Viral NA Small Volume Kit. Possible extraction bias was controlled by amplicon sequencing of mock communities. Fecal samples from canine, sheep, equine, bovine, and pig were thereafter subjected to the best performing DNA extraction method and shotgun metagenomic sequencing to determine sequencing efforts for functional and taxonomic analysis. ResultsThe four DNA extraction methods demonstrated similar community composition in the sequenced bacterial mock community. The QIAsymphony(R) PowerFecal(R) Pro DNA Kit was identified as the DNA extraction method of choice, and the resulting DNA was subjected to shotgun metagenomic sequencing with 20million PE reads. We found that higher number of reads increased the richness of observed genera between 100,000 and 5 million reads, after which higher sequencing effort did not increase the richness of the metagenomes. As for functional analysis, the number of low abundance functions in the metagenomes of the animals feces increased with sequencing depth above 20 million PE reads. ConclusionOur experiments identified several methods suitable for extracting DNA from feces collected on blood spot cards. The Qiagens Blood and Tissue kit on the QiaSymphony platform fulfilled the criteria of high yield, quality, and unbiased DNA, while maintaining high throughput for shotgun metagenomic sequencing. A sequencing depth of 20 million PE reads proved adequate for taxonomic estimations and identifying common functional pathways. Detecting rarer traits, however, requires more sequencing effort.