Open AccessAccurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels
Author(s) -
Caterina Manzari,
Annarita Oranger,
Bruno Fosso,
Elisabetta Piancone,
Graziano Pesole,
Anna Maria D’Erchia
Publication year - 2020
Publication title -
microbial genomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.476
H-Index - 28
ISSN - 2057-5858
DOI - 10.1099/mgen.0.000417
Subject(s) - metagenomics , biology , 16s ribosomal rna , dna , computational biology , microbiome , digital polymerase chain reaction , polymerase chain reaction , gene , genetics
The quantification of the total microbial content in metagenomic samples is critical for investigating the interplay between the microbiome and its host, as well as for assessing the accuracy and precision of the relative microbial composition which can be strongly biased in low microbial biomass samples. In the present study, we demonstrate that digital droplet PCR (ddPCR) can provide accurate quantification of the total copy number of the 16S rRNA gene, the gene usually exploited for assessing total bacterial abundance in metagenomic DNA samples. Notably, using DNA templates with different integrity levels, as measured by the DNA integrity number (DIN), we demonstrated that 16S rRNA copy number quantification is strongly affected by DNA quality and determined a precise correlation between quantification underestimation and DNA degradation levels. Therefore, we propose an input DNA mass correction, according to the observed DIN value, which could prevent inaccurate quantification of 16S copy number in degraded metagenomic DNAs. Our results highlight that a preliminary evaluation of the metagenomic DNA integrity should be considered before performing metagenomic analyses of different samples, both for the assessment of the reliability of observed differential abundances in different conditions and to obtain significant functional insights.