Comparison of targeted metagenomics and IS-Pro methods for analysing the lung microbiome

Study information

Reviewed Marked as Reviewed by Svetlana up on 2024-11-8

study design

cross-sectional observational, not case-control

Citation

PMID PubMed identifier for scientific articles.

34407769

DOI Digital object identifier for electronic documents.

10.1186/s12866-021-02288-x

URI

Authors

Goolam Mahomed T, Peters R, Pretorius G, Goolam Mahomed A, Ueckermann V, Kock MM, Ehlers MM

Journal

BMC microbiology

Year

2021

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 IS-Pro 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 IS-Pro 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.

Experiment 1

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Reviewed Marked as Reviewed by Svetlana up on 2024-11-8

Curated date: 2024/10/26

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Subjects

Location of subjects: South Africa

Host species Species from which microbiome was sampled. Contact us to have more species added.: Homo sapiens

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology: Sputum

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology

Group 0 name Corresponds to the control (unexposed) group for case-control studies: Samples sequenced by IS-pro method

Group 1 name Corresponds to the case (exposed) group for case-control studies: Samples sequenced by targeted metagenomics

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group: Targeted metagenomics is a method that focuses on analyzing specific regions of the microbial genome, typically V1-V3 regions of the 16s rRNA gene, to study the composition and diversity of microbial communities. While the IS-Pro method targets the intergenic spacer (IS) region between the 16 S rRNA and 23 S rRNA.

Group 0 sample size Number of subjects in the control (unexposed) group: 23

Group 1 sample size Number of subjects in the case (exposed) group: 23

Antibiotics exclusion Number of days without antibiotics usage (if applicable) and other antibiotics-related criteria used to exclude participants (if any): 1 month

Lab analysis

Sequencing type: 16S

16S variable region One or more hypervariable region(s) of the bacterial 16S gene: V1-V3

Sequencing platform Manufacturer and experimental platform used for quantifying microbial abundance: Illumina

Statistical Analysis

Data transformation Data transformation applied to microbial abundance measurements prior to differential abundance testing (if any).: raw counts

Statistical test: DESeq2

Significance threshold p-value or FDR threshold used for differential abundance testing (if any): 0.01

MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?: Yes

Alpha Diversity

Shannon Estimator of species richness and species evenness: more weight on species richness: increased

Simpson Estimator of species richness and species evenness: more weight on species evenness: unchanged

Signature 1

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Curated date: 2024/10/26

Curator: KateRasheed

Revision editor(s): KateRasheed, Svetlana up, WikiWorks

Source: Fig. 3

Description: Differential abundance in the sputum microbiota of Chronic obstructive pulmonary disease (COPD) patients between IS Pro and targeted metagenomics methods.

Abundance in Group 1: increased abundance in