Abstract
Here, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.
Highlights
The rapidly growing world population has intensified poultry production, which is predicted to produce about 130 million tons of chicken meat by 2020 according to the OECD/FAO reports[1]
A detailed assemblage of the different metagenomic isolation methods including important technical variables is shown in Supplementary Fig. S1
Our study did not evaluate the impact of alternative stool collection procedures with regards to starting volume, nucleic acid stabilizers, transportation, etc. and the effects of commonly used storage preservatives
Summary
The rapidly growing world population has intensified poultry production, which is predicted to produce about 130 million tons of chicken meat by 2020 according to the OECD/FAO reports[1]. A number of metagenomic studies rely on a wide range of commercially available kits[13,14]. Sample homogenization, bacterial lysis and DNA www.nature.com/scientificreports purification techniques are important sources of technical variations, which can significantly distort the apparent composition, structure and diversity of the microbiota[11,13,14,15,16]. The disruption of the bacteria with rigid cell walls and different membrane structures affects the effectiveness of polymerase chain reaction (PCR)-mediated metagenomic approaches, the choice of the method affects the quantity, quality, distribution and diversity of nucleic acid molecules[21,22,23,24,25]
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