Dataset Information

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa.

ABSTRACT: QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, United States) and RBB + C (Yu and Morrison, 2004) methodologies are widely employed to extract microbial DNA from rumen samples and can exhibit different efficiencies of obtaining DNA yield, quality, and downstream amplicon sequence analysis. No study has conducted to investigate the contributions of chemical and mechanical lysis on DNA extraction, which included chemical lysis from QIAamp Fast DNA Stool Mini Kit (QIA) and RBB + C (YM), bead (BB), and sand beating (SB). Effects of chemical lysis and bead beating (BB) were investigated by conducting a 2 × 2 factorial-designed experiment with four methodologies, including QIA without (QIA-) and with BB (QIA + BB), and YM without (YM-) and with BB (YM + BB). Comparisons between bead and sand were conducted by comparing methodologies of YM + BB and YM + SB. Comparing with QIA, YM had lower (P ? 0.10) OD₂₆₀ _/ ₂₈₀ and diversity of ZOTUs and length polymorphism of protozoal amplicons but harvested greater (P ? 0.086) DNA from fibrolytic bacteria such as Ruminococcaceae lineages. Including BB increased (P = 0.001) total DNA yield without affecting (P ? 0.55) OD₂₆₀ _/ ₂₈₀ and richness of bacterial ZOTUs but decreased (P ? 0.08) richness of both ZOTUs and length polymorphism of protozoal amplicon. Bead beating and SB showed no difference (P ? 0.19) in DNA yield and quality and bacterial and protozoal community. In summary, chemical lysis provided by RBB + C and QIAamp Fast DNA Stool Mini Kit should be better to extract DNA for analyzing bacterial and protozoal community, respectively. Sand can be an alternative beater for DNA extraction, and mechanical lysis is not recommended for protozoal community analysis.

SUBMITTER: Ma ZY

PROVIDER: S-EPMC7701101 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa.

Ma Zhi Yuan ZY Zhang Xiu Min XM Wang Rong R Wang Min M Liu Ting T Tan Zhi Liang ZL

Frontiers in microbiology 20201116

QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, United States) and RBB + C (Yu and Morrison, 2004) methodologies are widely employed to extract microbial DNA from rumen samples and can exhibit different efficiencies of obtaining DNA yield, quality, and downstream amplicon sequence analysis. No study has conducted to investigate the contributions of chemical and mechanical lysis on DNA extraction, which included chemical lysis from QIAamp Fast DNA Stool Mini Kit (QIA) and RBB + C (YM), bead ...[more]

PMID: 33304329

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Project description:ObjectiveRuminants are completely dependent on their microbiota for rumen fermentation, feed digestion, and consequently, their metabolism for productivity. This study aimed to evaluate the rumen bacteria of lactating yaks with different milk protein yields, using high-throughput sequencing technology, in order to understand the influence of these bacteria on milk production.MethodsYaks with similar high milk protein yield (high milk yield and high milk protein content, HH; n = 12) and low milk protein yield (low milk yield and low milk protein content, LL; n = 12) were randomly selected from 57 mid-lactation yaks. Ruminal contents were collected using an oral stomach tube from the 24 yaks selected. High-throughput sequencing of bacterial 16S rRNA gene was used.ResultsRuminal ammonia N, total volatile fatty acids, acetate, propionate, and isobutyrate concentrations were found to be higher in HH than LL yaks. Community richness (Chao 1 index) and diversity indices (Shannon index) of rumen microbiota were higher in LL than HH yaks. Relative abundances of the Bacteroidetes and Tenericutes phyla in the rumen fluid were significantly increased in HH than LL yaks, but significantly decreased for Firmicutes. Relative abundances of the Succiniclasticum, Butyrivibrio 2, Prevotella 1, and Prevotellaceae UCG-001 genera in the rumen fluid of HH yaks was significantly increased, but significantly decreased for Christensenellaceae R-7 group and Coprococcus 1. Principal coordinates analysis on unweighted UniFrac distances revealed that the bacterial community structure of rumen differed between yaks with high and low milk protein yields. Furthermore, rumen microbiota were functionally enriched in relation to transporters, ABC transporters, ribosome, and urine metabolism, and also significantly altered in HH and LL yaks.ConclusionWe observed significant differences in the composition, diversity, fermentation product concentrations, and function of ruminal microorganisms between yaks with high and low milk protein yields, suggesting the potential influence of rumen microbiota on milk protein yield in yaks. A deeper understanding of this process may allow future modulation of the rumen microbiome for improved agricultural yield through bacterial community design.

Dataset Information

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa.

Publications

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa.

Similar Datasets

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