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.

Fig 1. Experimental design.

A total of 72 four-week-old SPF chickens were divided into three groups on the day of sampling. Samples were collected from individual birds and pooled as shown above. Each sample pool was aliquoted into several replicates that were evenly divided between the BT and ZB kits. LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.

Fig 2. Comparison of quantity and quality of DNA from samples extracted using the ZB and BT kits.

The elution volume was standardized to 50 μL for both kits. DNA quality was expressed as ratios of absorbances at (A) 260nm and 280nm wavelengths (A260/A280) and (B) 260nm and 230nm wavelengths (A260/A230). (C) DNA yield was expressed as mass per unit volume (ng/μL). Statistical differences, * p < 0.05 (pairwise Kruskal-Wallis tests, with a Benjamini-Hochberg correction for false discovery). LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.

Fig 3. Between-kit comparison of pre- and post-sequencing 16S rRNA gene copies for each sample type.

(A) Pre-sequencing 16S rRNA gene copies per nanogram of total DNA. Quantitative PCR was used to determine the number of 16S rRNA gene copies per μL of sample. The DNA samples were then normalized to 1.67 × 10⁵ 16S rRNA gene copies/μL for subsequent PCR prior to sequencing. (B) Post-sequencing 16S rRNA gene sequence counts. The sequences were quality-filtered as described in Materials and methods. Statistical differences, * p < 0.05 (pairwise Kruskal-Wallis tests, with a Benjamini-Hochberg correction for false discovery). LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.

Fig 4. Host sequences detected in Illumina sequencing data.

(A) Sequence length distribution showing the size ranges for avian mitochondria (175, 176, 178, and 194 bp) and 16S rRNA gene sequences (250–264 bp). (B) Statistical comparisons of mitochondria gene sequences in different tissue types. Statistical differences, * p < 0.05 (pairwise Kruskal-Wallis tests, with a Benjamini-Hochberg correction for false discovery).

Fig 5. Comparison of relative abundances of different bacteria classes in samples extracted using the BT and ZB kits.

LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.

Fig 6. Comparison of alpha diversity metrics from samples extracted using the ZB and BT kits.

(A) Species richness was estimated based on the number of observed amplicon sequence variants (ASVs) in each sample. (B) Species evenness in each sample was estimated using Pielou’s evenness index. Statistical differences, * p < 0.05 (pairwise Kruskal-Wallis tests, with a Benjamini-Hochberg correction for false discovery). LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.

Fig 7. Comparison of beta diversity metrics from samples extracted using the BT and ZB kits.

Unweighted (A) and Weighted (B) UniFrac distances were compared between samples from a given sample type. No statistical differences were found between the kits (pairwise permutational multivariate analysis of variance, PERMANOVA, with a Benjamini-Hochberg correction for false discovery). LCS, live choanal swab; ECS, euthanized choanal swab; LTS, live tracheal swab; ETS, euthanized tracheal swab; NC, nasal cavity wash; UT, upper trachea wash; LT, lower trachea wash; LRV, lower respiratory lavage.