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

Edit History Delete

Flag for review

Your review change was submittedDuplicate this Experiment Add a new Signature

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

Curated date: 2024/10/26

Curator: Tosin

Revision editor(s): Tosin, Svetlana up

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 Expectoration,Sputum,sputum

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology: Chronic obstructive pulmonary disease Airflow Obstruction, Chronic,Airflow Obstructions, Chronic,CAFL - Chronic airflow limitation,CAL - Chronic airflow limitation,CAO - Chronic airflow obstruction,Chronic airflow limitation,Chronic Airflow Obstruction,Chronic Airflow Obstructions,Chronic airway disease,Chronic airway obstruction,chronic airway obstruction, NEC in ICD9CM_2006,chronic airway obstruction, not elsewhere classified,Chronic irreversible airway obstruction,CHRONIC OBSTRUCTIVE AIRWAY DIS,chronic obstructive airway disease,Chronic Obstructive Airways Disease,chronic obstructive airways disease,chronic obstructive airways disease NOS,chronic obstructive airways disease NOS (disorder),CHRONIC OBSTRUCTIVE LUNG DIS,Chronic Obstructive Lung Disease,chronic obstructive lung disease,chronic obstructive lung disease (disorder),chronic obstructive lung disease [Ambiguous],Chronic obstructive lung disease, NEC,Chronic obstructive lung disease, NOS,CHRONIC OBSTRUCTIVE PULM DIS,chronic obstructive pulmonary disease,Chronic Obstructive Pulmonary Disease (COPD),chronic obstructive pulmonary disease (COPD),chronic obstructive pulmonary disease and allied conditions,Chronic obstructive pulmonary disease finding,Chronic obstructive pulmonary disease finding (finding),Chronic obstructive pulmonary disease NOS,CHRONIC OBSTRUCTIVE PULMONARY DISEASE, (COPD),chronic obstructive pulmonary disease, (COPD),COAD,COAD - Chronic obstructive airways disease,COLD,cold,COLD (chronic obstructive lung disease),cold (chronic obstructive lung disease),COLD - Chronic obstructive lung disease,COPD,COPD - Chronic obstructive pulmonary disease,COPD NOS,COPD, CHRONIC OBSTRUCTIVE PULMONARY DISEASE,COPD, chronic obstructive pulmonary disease,DISEASE (COPD), CHRONIC OBSTRUCTIVE,disease (COPD), chronic obstructive,Dops,obstructive lung disease, chronic,OBSTRUCTIVE PULMONARY DISEASE (COPD), CHRONIC,obstructive pulmonary disease (COPD), chronic,PULM DIS CHRONIC OBSTRUCTIVE,PULMONARY DISEASE (COPD), CHRONIC OBSTRUCTIVE,pulmonary disease (COPD), chronic obstructive,Pulmonary Disease, Chronic Obstructive,Chronic obstructive pulmonary disease

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

Edit History Delete

Flag for review

Your review change was submitted

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

Curated date: 2024/10/26

Curator: KateRasheed

Revision editor(s): KateRasheed, Svetlana up

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 Samples sequenced by targeted metagenomics

NCBI	Quality Control	Links
Actinomyces
Granulicatella
Leptotrichia
Oleomonas
Veillonella

Revision editor(s): KateRasheed, Svetlana up

Signature 2

Edit History Delete

Flag for review

Your review change was submitted

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

Curated date: 2024/10/27

Curator: Aleru Divine

Revision editor(s): Aleru Divine

Source: Figure 3

Description: Differential abundance in the sputum microbiota of COPD patients between IS Pro and targeted metagenomics methods.

Abundance in Group 1: decreased abundance in Samples sequenced by targeted metagenomics

NCBI	Quality Control	Links
Corynebacterium
Parvimonas
Serratia
Pseudomonas
Lactobacillus
Fusobacterium
Peptostreptococcus
Eikenella
Escherichia
Micrococcus
Proteus
Neisseria
Burkholderia

Revision editor(s): Aleru Divine