Computational protocol: Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

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Protocol publication

[…] 16S rDNA sequencing was carried out using the Illumina MiSeq platform. Such sequencing is amplicon-based, targeting the variable region V3-V4 of the 16S rDNA gene and generates PCR products with a length of ~460 bp. Paired-end, 300 × 2 bp sequencing was carried out to cover the whole PCR product from the two opposite ends. For library construction, the Illumina official guide for 16S rDNA sequencing library preparation was used as reference. Bioinformatics analysis was carried out using the 16S Metagenomics app v1.0.1 provided in the Illumina BaseSpace online platform, including reads quality control, reads alignment, assembly and annotations. For taxonomic classification of the 16S rDNA reads, the Ribosomal Database Project (RDP) Classifier was used for classification (Wang et al., ) and an Illumina-curated version of the GreenGenes taxonomic database was used as reference database (DeSantis et al., ). Both applications were inbuilt in BaseSpace. […]

Pipeline specifications

Software tools BaseSpace, RDP Classifier
Databases Greengenes
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Glucosephosphate Dehydrogenase Deficiency
Chemicals Chloroform