The objective of Metagenomics is to study the composition of the microbial communities sampled from their natural environment. It involves the analysis of the hypervariable regions of the 16s rRNA gene sequences which expresses major sequence diversity among different microorganisms. The sequences are clustered against the reference sequence collection and each of the resulting clusters, known as Operational Taxonomic Unit (OTU) represents a taxonomic unit of the microbial species or genus based on the sequence similarity threshold. The clustering method results in an OTU table, listing the abundances of OTUs in the samples under examination. Further analysis includes evaluation of beta and alpha diversities of the samples.
Eurofins genomics provides 16S amplicon on Illumina MiSeq sequencing platform for V3-V4 regions and also does for customized amplicon such as archaea, cyanobacteria and fungal samples.
Bioinformatics workflow for 16S MicroBiome Profiling analysis:
Quality check of raw reads:
The raw reads will be subjected to quality filtration and adapter trimming using Trimmomatic software. The high quality data will be used for downstream analysis.
The paired end reads are stitched/merged together using FLASH tool in order to cover the complete 16s rRNA “hypervariable” region.
QIIME (Quantitative Insights Into Microbial Ecology) tool is used for OTU picking by clustering all the reads against the Greengene reference database. The clustering process is carried out using the UCLUST algorithm. The output is a biom file listing the abundances of OTUs for each sample.
QIIME tool assigns taxonomy to the representative of each OTU by comparing to the Greengene taxonomy reference database. It thus provides taxonomic classification for the OTUs from Kingdom to Species level.
Alpha Diversity is an estimation of number of species in each sample and is represented through the rarefaction plot.
This includes calculation of beta diversity which estimates the differences in species diversity between samples which is represented through a PCoA plot.
- Quality filtration of data,
- Phylogenetic analysis of microbial distribution
- Heat map, abundance and identification of microbial community
- Rank abundance plot
- Analysis for dominant population
- Taxa identification
- LCA plot
- Rarefraction curve
- Alpha diversity
- Comparative analysis and beta diversity (PCoA Plot)
- Krona Chart
- Comprehensive report with publication standard methodology, graphs and tables.