Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling.
Sci Rep
; 10(1): 3419, 2020 02 25.
Article
in En
| MEDLINE
| ID: mdl-32099013
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA, Bacterial
/
Chickens
/
Metagenome
/
Metagenomics
/
Gastrointestinal Microbiome
/
Gram-Negative Bacteria
Limits:
Animals
Language:
En
Journal:
Sci Rep
Year:
2020
Document type:
Article
Affiliation country:
Hungary