Format

Send to

Choose Destination
Microbiome. 2018 Sep 10;6(1):155. doi: 10.1186/s40168-018-0543-z.

Quantification of variation and the impact of biomass in targeted 16S rRNA gene sequencing studies.

Author information

1
USC Keck School of Medicine and Children's Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA, 90027, USA.
2
Children's Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA, 90027, USA.
3
David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA, 90095, USA.
4
USC Keck School of Medicine, 1975 Zonal Ave, Los Angeles, CA, 90033, USA.
5
David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA, 90095, USA. gracea@mac.com.

Abstract

BACKGROUND:

Recent advances in sequencing technologies and bioinformatics tools have allowed for large-scale microbiome studies that are rapidly advancing medical research. However, small changes in technique or analysis can significantly alter the results and lead to conflicting findings. Quantifying the technical versus biological variation expected in targeted 16S rRNA gene sequencing studies and how this variation changes with input biomass is critical to guide meaningful interpretation of the current literature and plan future research.

RESULTS:

Data were compiled from 469 sequencing libraries across 19 separate targeted 16S rRNA gene sequencing runs over a 2.5-year time period. Following removal of contaminant sequences identified from negative controls, 244 samples retained sufficient reads for further analysis. Coefficients of variation for intra- and inter-assay variation from repeated measurements of a bacterial mock community ranged from 8.7 to 37.6% (intra) and 15.6 to 80.5% (inter) for all but one genus of bacteria whose relative abundance was greater than 1%. Intra- versus inter-assay Bray-Curtis pairwise distances for a single stool sample were 0.11 versus 0.31, whereas intra-assay variation from repeat stool samples from the same donor was greater at 0.38 (Wilcoxon p = 0.001). A dilution series of the bacterial mock community was used to assess the effect of input biomass on variability. Pairwise distances increased with more dilute samples, and estimates of relative abundance became unreliable below approximately 100 copies of the 16S rRNA gene per microliter. Using this data, we created a prediction model to estimate the expected variation in microbiome measurements for given input biomass and relative abundance values.

CONCLUSIONS:

Well-controlled microbiome studies are sufficiently robust to capture small biological effects and can achieve levels of variability consistent with clinical assays. Relative abundance is negatively associated with measures of variability and has a stronger effect on variability than does absolute biomass, suggesting that it is feasible to detect differences in bacterial populations in very low-biomass samples. Further, by quantifying the effect of biomass and relative abundance on compositional variability, we developed a tool for defining the expected variance in a given microbiome study.

KEYWORDS:

Accuracy; Biological variation; Biomass; Precision; Technical variation

PMID:
30201048
PMCID:
PMC6131952
DOI:
10.1186/s40168-018-0543-z
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for BioMed Central Icon for PubMed Central
Loading ...
Support Center