Abstract
Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.
Highlights
The mean value for the lower respiratory lavage samples extracted with the ZymoBIOMICS DNA Miniprep (ZB) kit was higher than 2.0 which was significantly higher compared to the blood and tissue (BT) kit (Fig 2A)
Even though disparities in the relative abundance of 16S rRNA gene sequences were detected between the kits, only Listeria monocytogenes and Staphylococcus aureus, both grampositive bacteria, were found to be significantly higher in the ZymoBIOMICS DNA Miniprep kit (ZB kit) as detected using DESeq 2 [47]
Using the Blood and Tissue kit (BT kit) in our previous studies, we were able to characterize the microbial communities present in sinus cavities, tracheas and lower respiratory tracts of turkeys, chicken broilers, and chicken layers raised in commercial settings, and specific-pathogen free chickens raised for research [2, 15,16,17]
Summary
Enzymatic and chemical lysis methods are less likely to damage DNA, but their inefficiency in disrupting certain types of bacteria may introduce bias in the biodiversity profiles [22] Another important consideration for choosing extraction kits are the contaminating DNAs that are ubiquitously present in kits and reagents. Five different DNA extraction kits were previously used in poultry respiratory microbiota studies, including BiOstic FFPE Tissue DNA Isolation Kit, PowerSoil DNA Isolation Kit, QIAmp DNA Mini Kit, Maxwell RSC PureFood Pathogen Kit, and Qiagen DNeasy Blood and Tissue Kit [2, 15,16,17, 25,26,27,28] These kits typically employ a combination of mechanical, chemical, and enzymatic disruption methods to increase DNA yields. After sequencing of normalized samples, both kits produced similar microbiome data in terms of bacterial diversity at the amplicon sequence variant (ASV) level and taxonomic profiles at the class and genus levels
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.