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Comparison of targeted metagenomics and IS-Pro methods for analysing the lung microbiome
Goolam Mahomed, Tanweer; Peters, Remco P.H.; Pretorius, G.H.J.; Mahomed, A. Goolam; Ueckermann, Veronica; Kock, Marleen M.; Ehlers, Marthie Magdaleen
Date:
2021-08-18
Abstract:
BACKGROUND : Targeted metagenomics and IS-Pro method are two of the many methods that have been used to
study the microbiome. The two methods target different regions of the 16 S rRNA gene. The aim of this study was
to compare targeted metagenomics and IS-Pro methods for the ability to discern the microbial composition of the
lung microbiome of COPD patients.
METHODS : Spontaneously expectorated sputum specimens were collected from COPD patients. Bacterial DNA was
extracted and used for targeted metagenomics and IS-Pro method. The analysis was performed using QIIME2
(targeted metagenomics) and IS-Pro software (IS-Pro method). Additionally, a laboratory cost per isolate and time
analysis was performed for each method.
RESULTS : Statistically significant differences were observed in alpha diversity when targeted metagenomics and ISPro
methods’ data were compared using the Shannon diversity measure (p-value = 0.0006) but not with the
Simpson diversity measure (p-value = 0.84). Distinct clusters with no overlap between the two technologies were
observed for beta diversity. Targeted metagenomics had a lower relative abundance of phyla, such as the
Proteobacteria, and higher relative abundance of phyla, such as Firmicutes when compared to the IS-Pro method.
Haemophilus, Prevotella and Streptococcus were most prevalent genera across both methods. Targeted
metagenomics classified 23 % (144/631) of OTUs to a species level, whereas IS-Pro method classified 86 % (55/64) of
OTUs to a species level. However, unclassified OTUs accounted for a higher relative abundance when using the ISPro
method (35 %) compared to targeted metagenomics (5 %). The two methods performed comparably in terms
of cost and time; however, the IS-Pro method was more user-friendly.
CONCLUSIONS : It is essential to understand the value of different methods for characterisation of the microbiome.
Targeted metagenomics and IS-Pro methods showed differences in ability in identifying and characterising OTUs,
diversity and microbial composition of the lung microbiome. The IS-Pro method might miss relevant species and
could inflate the abundance of Proteobacteria. However, the IS-Pro kit identified most of the important lung pathogens, such as Burkholderia and Pseudomonas and may work in a more diagnostics-orientated setting. Both
methods were comparable in terms of cost and time; however, the IS-Pro method was easier to use.
Description:
SUPPLEMENTARY MATERIAL: Table S1. Inclusion and exclusion criteria for COPD patients in this study. Table S2. Clinical characteristic of patients. Table S3. Comparison of the number of amplicons and operational taxonomic units for each sample for the targeted metagenomics and IS-Pro methods. Figure S1. Relative abundance of specific phyla in the sputum microbiome of COPD participants as detected by targeted metagenomics and IS-Pro methods (n = 23). The dots represent the different abundances of each sample, according to the different phyla. Phyla that are depicted with a single line on the y-axis were not present in any samples for that method. Figure S2. Bar plots showing the relative abundance of genera in the sputum microbiome of COPD participants as characterised by targeted metagenomics and IS-Pro methods (n = 23). The operational taxonomic units that could not be classified at a genus level are indicated as NA on the graph. Figure S3. The distribution of the unclassified operational taxonomic units (OTUs) at a class level of the sputum microbiome of COPD participants for targeted metagenomics and IS-Pro methods by phyla. At a class level, all the OTUs from targeted metagenomics could be classified.
