NGS samples were prepared according to the 16S Metagenomic Sequencing Library Preparation guide (part# 15044223 rev A; Illumina, San Diego, CA). The PCR amplicon was obtained using primers (containing overhang adapters) as previously described. 27, 28 Libraries were quantified through PicoGreen dsDNA Quantitation Assay (ThermoFisher Scientific, Waltham, MA) and validated on Bioanalyzer DNA 1000 chip (Agilent, Santa Clara, CA) for the presence of the expected amplicon. Sequencing was performed on a MiSeq desktop sequencer (Illumina). The sequence reads were analyzed in the cloud environment BaseSpace through the 16S Metagenomics app (version 1.0.1; Illumina): the taxonomic database used was an Illumina-curated version of the May 2013 release of the Greengenes Consortium Database (greengenes.secondgenome .com); the classification algorithm was an implementation of the Ribosomal Database Project Classifier. 29 The Principal Coordinates Analysis was generated through the 16S Metagenomics app using classic multidimensional scaling on a Pearson covariance distance matrix created from per-sample normalized classification abundance vectors. Detection cutoff was set at 1%. NGS analysis was performed on a subset of positive samples (n = 48), whenever enough DNA was available for this additional experiment.
16s metagenomics app
Manufactured by Illumina
The 16S Metagenomics app is a software tool that enables the analysis of microbial communities by sequencing the 16S ribosomal RNA gene. It provides a standardized workflow for processing and interpreting 16S rRNA sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Basespace, by Illumina (2 mentions)
Fastq toolkit, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Spss software version 22, by IBM (1 mentions)
Picogreen dsdna quantitation assay, by Thermo Fisher Scientific (1 mentions)
16s metagenomic sequencing library preparation guide, by Illumina (1 mentions)
Bioanalyzer dna 1000 chip, by Agilent Technologies (1 mentions)
Miseq desktop sequencer, by Illumina (1 mentions)
6 protocols using 16s metagenomics app
1
16S Metagenomics Sequencing of Microbial Communities
2
16S rRNA Taxonomic Classification
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The primer sequences were removed, and reads with low quality scores (average score < 20) were filtered out using the FASTQ Toolkit within BaseSpace (Illumina, Version 2.2.0). The 16S Metagenomics app within BaseSpace (Version 1.0.1) was used to perform a taxonomic classification, using an Illumina-curated taxonomic database RefSeq RDP 16S v3 [12 (link)] and the RDP naive Bayes taxonomic classification algorithm with an accuracy of >98.2% at the species level [13 (link)]. Default parameters were used for all software unless otherwise noted.
3
16S Bacterial Microbiome Assessment
4
Metagenomic Analysis of Microbial Diversity
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One-way analysis of variances (ANOVA) was performed for multiple data using IBM SPSS software (version 22) to check for quantitative differences between samples. Data were significantly different at p = 0.05.
Sequencing data obtained from metagenomic analysis were analysed using the 16S Metagenomics App in Illumina Basespace (San Diego, California). Sequences were quality trimmed, filtered and processed using the Quantitative Insights Into Microbial Ecology (QIIME) package available from the Illumina Basespace website [33 (link)]. The obtained sequencing data from the QIIME were further analysed by MEGAN 6 [34 (link)].
An Operational Taxonomic Units (OTU) genus-level table was imported into Primer 7 software; the data were standardised and transformed using square root. Shannon diversity and richness indices were also calculated using Primer 7 software, as described in the software manual. The standardised and transformed data were subjected to resemblance analysis using S17 Bray Curtis similarity resemblance measure and cluster analysis applied to generate plot dendrograms using the group average cluster mode. In addition, principal coordinate analysis (PCoA) was performed using Primer 7 [35 ].
Sequencing data obtained from metagenomic analysis were analysed using the 16S Metagenomics App in Illumina Basespace (San Diego, California). Sequences were quality trimmed, filtered and processed using the Quantitative Insights Into Microbial Ecology (QIIME) package available from the Illumina Basespace website [33 (link)]. The obtained sequencing data from the QIIME were further analysed by MEGAN 6 [34 (link)].
An Operational Taxonomic Units (OTU) genus-level table was imported into Primer 7 software; the data were standardised and transformed using square root. Shannon diversity and richness indices were also calculated using Primer 7 software, as described in the software manual. The standardised and transformed data were subjected to resemblance analysis using S17 Bray Curtis similarity resemblance measure and cluster analysis applied to generate plot dendrograms using the group average cluster mode. In addition, principal coordinate analysis (PCoA) was performed using Primer 7 [35 ].
5
16S Metagenomics Analysis of Microbial Communities
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The analysis was applied using the 16S Metagenomics app (version 1.1.0) from the Illumina BaseSpace platform and sequences with Phred < Q20 were removed. Operational taxonomic units (OTUs) were created, using Ribosomal Database Project Classifier (Wang et al., 2007 (link)) against the Illumina-curated version of GreenGenes reference taxonomy database (DeSantis et al., 2006 (link)), as described previously (Yuan et al., 2018 (link)). The classified OTUs were defined at ≥97% of sequence homology and converted to percentages (relative abundances), to determine the representation of each microbe among treatments. Alpha diversity indexes were calculated using the EstimateS version 9.1.01 , by different metrics (Chao, Shannon, Simpson) after performing a rarefaction analysis. Rarefied OTU to 54,488 sequences (lowest number of reads obtained) were used to obtain these indices. Finally, all sequences were deposited to the National Centre for Biotechnology Information (NCBI) in Sequence Read Archive (SRA) under the BioProject PRJNA600153 .
6
Taxonomic Profiling of Microbial Communities
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Sequence data were analyzed using the 16S Metagenomics App provided by BaseSpace software (version 1.0.1, Illumina Inc.) which performs taxonomic classification based on the Greengenes database (available online: http://greengenes.secondgenome.com/downloads/database/13_5 ). For each sample, the relative abundance of the top eight taxonomic classifications at each level (from phylum to species) was considered for statistical analysis. Moreover, the software calculated the Shannon index (α-diversity) and the number of species (richness) found in each sample.
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