National Microbiology Laboratory
Bioinformatics section

Author Of 1 Presentation

Biomarkers and Bioinformatics Poster Presentation

P0171 - The gut mycobiome in pediatric multiple sclerosis: establishing a bioinformatics pipeline (ID 876)

Speakers
Presentation Number
P0171
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Studies examining the role of the microbiota in multiple sclerosis (MS) often focus on the gut bacteria; few have considered a potential role of gut mycobiota. Methods for evaluating gut mycobiota are lacking and require systematic evaluation of sequencing protocols, reference databases, and bioinformatics pipelines to properly investigate possible gut mycobiome influences on MS.

Objectives

We set out to evaluate the performance of different sequencing conditions and analytical approaches for characterizing the gut mycobiome in a cohort of healthy individuals and cases with monophasic acquired demyelinating syndrome (mono ADS) or pediatric-onset MS.

Methods

We first assessed a mock-community control pool of known, staggered quantities of 19 defined fungal organisms. We then assessed 201 stool samples obtained from our cohort of 52 healthy individuals, 49 individuals with mono ADS, and 46 participants with pediatric-onset MS. The fungal internal transcribed spacer (ITS) 2 region was sequenced using the Illumina® MiSeq platform. Varying concentrations of PhiX Control v3 Library spike-in were tested to address low-complexity amplicon sequencing. Generated sequences were characterized by the UNITE database—a curated collection of eukaryotic ITS sequences—in conjunction with three distinct fungal sequence analysis pipelines: LotuS, mothur, and PIPITS.

Results

Taxa identified in our mock-community differed across sequencing conditions but were similar between technical replicates. LotuS correctly classified 7 taxa at species-level, 7 taxa at genus-level, whereas 5 remained unclassified. Mothur correctly identified 5 species-level taxa, 11 genus-level taxa, whereas 3 remained unclassified. Lastly, PIPITS correctly identified only 3 species-level taxa, 12 genus-level, while 4 remained unclassified. We successfully generated sequence data for 112 of 147 (76%) individuals (70 females; 42 males). The mean age at stool sample collection was 17.3 (SD 5.1) years. Of the tested sequencing conditions, a spike-in of 50% PhiX produced the highest-quality reads.

Conclusions

The LotuS pipeline best identified fungal taxa in our mock-community, with optimal resolution to species level. Sequencing read quality was optimal when 50% PhiX was used for sequencing ITS2 amplicon libraries of stool samples. Establishment of this validated sequencing pipeline, confirmed using a mock-community with known fungal identities, will aid characterization of gut mycobiomes for our cohort of individuals with/without pediatric-onset MS.

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Presenter Of 1 Presentation

Biomarkers and Bioinformatics Poster Presentation

P0171 - The gut mycobiome in pediatric multiple sclerosis: establishing a bioinformatics pipeline (ID 876)

Speakers
Presentation Number
P0171
Presentation Topic
Biomarkers and Bioinformatics

Abstract

Background

Studies examining the role of the microbiota in multiple sclerosis (MS) often focus on the gut bacteria; few have considered a potential role of gut mycobiota. Methods for evaluating gut mycobiota are lacking and require systematic evaluation of sequencing protocols, reference databases, and bioinformatics pipelines to properly investigate possible gut mycobiome influences on MS.

Objectives

We set out to evaluate the performance of different sequencing conditions and analytical approaches for characterizing the gut mycobiome in a cohort of healthy individuals and cases with monophasic acquired demyelinating syndrome (mono ADS) or pediatric-onset MS.

Methods

We first assessed a mock-community control pool of known, staggered quantities of 19 defined fungal organisms. We then assessed 201 stool samples obtained from our cohort of 52 healthy individuals, 49 individuals with mono ADS, and 46 participants with pediatric-onset MS. The fungal internal transcribed spacer (ITS) 2 region was sequenced using the Illumina® MiSeq platform. Varying concentrations of PhiX Control v3 Library spike-in were tested to address low-complexity amplicon sequencing. Generated sequences were characterized by the UNITE database—a curated collection of eukaryotic ITS sequences—in conjunction with three distinct fungal sequence analysis pipelines: LotuS, mothur, and PIPITS.

Results

Taxa identified in our mock-community differed across sequencing conditions but were similar between technical replicates. LotuS correctly classified 7 taxa at species-level, 7 taxa at genus-level, whereas 5 remained unclassified. Mothur correctly identified 5 species-level taxa, 11 genus-level taxa, whereas 3 remained unclassified. Lastly, PIPITS correctly identified only 3 species-level taxa, 12 genus-level, while 4 remained unclassified. We successfully generated sequence data for 112 of 147 (76%) individuals (70 females; 42 males). The mean age at stool sample collection was 17.3 (SD 5.1) years. Of the tested sequencing conditions, a spike-in of 50% PhiX produced the highest-quality reads.

Conclusions

The LotuS pipeline best identified fungal taxa in our mock-community, with optimal resolution to species level. Sequencing read quality was optimal when 50% PhiX was used for sequencing ITS2 amplicon libraries of stool samples. Establishment of this validated sequencing pipeline, confirmed using a mock-community with known fungal identities, will aid characterization of gut mycobiomes for our cohort of individuals with/without pediatric-onset MS.

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