Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

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.

Development of an efficient antimicrobial susceptibility testing method with species identification by Nanopore sequencing of 16S rRNA amplicons

“…First, the difference in DNA extraction efficiency among different bacteria must be minimized. This condition could be satisfied using the ZymoBIOMICS DNA Miniprep Kit, which has been shown to be an effective extraction method for 16S rRNA amplicon analysis referred in several papers [ 28 , 29 ].…”

Standardization of SYBR Green-Based Real-Time PCR Through the Evaluation of Different Thresholds for Different Skin Layers: An Accuracy Study and Track of the Transmission Potential of Multibacillary and Paucibacillary Leprosy Patients

“…This result shows that although the separation of skin layers may not be extremely precise, this will have a limited influence on the PCR results. This result may be explained by the fact that modern extraction kits are efficient in isolating different sorts of biological samples ( Manta et al, 2020 ; Abundo et al, 2021 ). Although there is controversy related to blood samples ( Manta et al, 2020 ), previous studies have also not found significant differences in accuracy properties depending on different sample types when evaluating molecular biology techniques.…”

Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression

“…Our optimized methods for sequencing and sequence processing are detailed in our recent reports ( Abundo et al, 2021 ; Taylor et al, 2020 ). For each sample, DNA was quantified using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), adjusted to a concentration of 100 ng/µL, and shipped to the University of Minnesota Genomics Center (Minneapolis, MN, USA) for sequencing.…”

“…Avian influenza viruses are among the numerous pathogens that induce strong innate immune responses such as IFNs and pro-inflammatory cytokines ( Akira, Uematsu & Takeuchi, 2006 ; Medzhitov & Janeway Jr, 2000 ; Mogensen, 2009 ) which can trigger dysbiosis in the respiratory tract of mammals ( Duvigneau et al, 2016 ; Engelsoy, Rangel & Demirel, 2019 ; Madouri et al, 2018 ; Sharma-Chawla et al, 2019 ) and in the avian gut ( Ganz et al, 2017 ; Hird et al, 2018 ; Li et al, 2018 ; Yitbarek et al, 2018c ; Zhao et al, 2018 ). However, influenza virus-induced dysbiosis of avian respiratory microbiota has not been described due to challenges in respiratory sampling methodologies ( Abundo et al, 2020 ; Abundo et al, 2021 ). Furthermore, there is a lack of information about the impacts of influenza virus infection on turkey microbiota despite turkeys generally being highly susceptible to diverse influenza virus strains ( Pantin-Jackwood et al, 2017 ; Pillai et al, 2010b ; Tumpey, Kapczynski & Swayne, 2004 ).…”