Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples
Por:
Pérez-Brocal V, Magne F, Ruiz-Ruiz S, Ponce CA, Bustamante R, Martin VS, Gutierrez M, Gatti G, Vargas SL and Moya A
Publicada:
15 oct 2020
Ahead of Print:
15 oct 2020
Categoría:
Multidisciplinary
Resumen:
Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.
Filiaciones:
:
Department of Genomics and Health, Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO-Public Health), Avda. Cataluña 21, 46020, València, Spain
CIBER in Epidemiology and Public Health (CIBERESP), Madrid, Spain
Magne F:
Microbiology and Mycology Program, Biomedical Sciences Institute (ICBM), University of Chile School of Medicine, Av. Independencia 1027, Independencia, Santiago, Chile.
:
Department of Genomics and Health, Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO-Public Health), Avda. Cataluña 21, 46020, València, Spain
CIBER in Epidemiology and Public Health (CIBERESP), Madrid, Spain
Ponce CA:
Microbiology and Mycology Program, Biomedical Sciences Institute (ICBM), University of Chile School of Medicine, Av. Independencia 1027, Independencia, Santiago, Chile
Bustamante R:
Microbiology and Mycology Program, Biomedical Sciences Institute (ICBM), University of Chile School of Medicine, Av. Independencia 1027, Independencia, Santiago, Chile
Martin VS:
Médico Legal Institute of Chile, Santiago, Chile
Gutierrez M:
Médico Legal Institute of Chile, Santiago, Chile
Gatti G:
Médico Legal Institute of Chile, Santiago, Chile
:
Department of Genomics and Health, Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO-Public Health), Avda. Cataluña 21, 46020, València, Spain.
CIBER in Epidemiology and Public Health (CIBEResp), Madrid, Spain.
Institute for Integrative Systems Biology (I2SysBio), University of Valencia and Spanish National Research Council (CSIC), València, Spain.
gold, Green Published
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