Computational protocol: Correlation of placental microbiota with fetal macrosomia and clinical characteristics in mothers and newborns

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Protocol publication

[…] Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using QuantiFluor™-ST (Promega BioSciences LLC, Sunnyvale, CA, USA) according to the standard protocols. Then, purified amplicons were pooled and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. All raw reads were screened according to barcode and primer sequences, using Quantitative Insights Into Microbial Ecology (QIIME, version 1.17), with the following criteria: 1) The 300 bp reads were truncated at any site receiving an average quality score <20 over a 10 bp sliding window; 2) the truncated reads that were <50 bp were abandoned; 3) Sequences that overlap shorter than 10 bp, or reads containing ambiguous characters, or >2 nucleotide mismatch in primer matching were removed. Operational taxonomic units (OTUs) were clustered with the cut-off of 97% similarity using UPARSE (version 7.1, http://drive5.com/uparse/), and UCHIME was utilized to identify and remove chimeric sequences. RDP Classifier (http://rdp.cme.msu.edu/) was used to analyze the phylogenetic affiliation of each 16S rRNA gene sequence, against the silva (SSU115) 16S rRNA database with confidence threshold of 70% []. Weighted UniFrac distance metrics analysis was performed using OTUs for each sample, and principal coordinates analysis (PCoA) analysis was conducted according to the matrix of distance []. Linear discriminant analysis (LDA) effect size (LEfSe) was used to elucidate the differences of bacterial taxa. The LDA score ≥2 was considered to be important contributors to the model. The cladogram was drawn using the Huttenhower Galaxy web application (The Huttenhower Lab, Boston, MA, USA) via the LEfSe algorithm (http://huttenhower.sph.harvard.edu/lefse/) []. […]

Pipeline specifications

Software tools QIIME, UPARSE, UCHIME, RDP Classifier, UniFrac, LEfSe
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Metabolic Diseases