16S/18S/ITS MicroBiome Profiling Analysis

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 and ITS/18S 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 bacterial (V3-V4 region), ITS/18S amplicon on Illumina MiSeq sequencing platform and also provides customized amplicon such as archaea, cyanobacteria etc.

Workflow and deliverables

Bioinformatics workflow for 16S MicroBiome Profiling analysis:

a. Quality check of raw reads:

The raw reads will be subjected to quality filtration and adapter trimming. The high quality data will be used for downstream analysis. The paired end reads are stitched/merged together in order to cover the complete 16s rRNA “hypervariable” region/ ITS/18S region.

b. OTU Picking

QIIME (Quantitative Insights Into Microbial Ecology) tool is used for OTU picking by clustering all the reads against the Greengene/Silva/Unite 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.

c. Taxonomy Assignment

QIIME tool assigns taxonomy to the representative of each OTU by comparing to the taxonomy reference database. It thus provides taxonomic classification for the OTUs from Kingdom to Species level.

d. Sample Analysis

Alpha Diversity is an estimation of number of species in each sample and is represented through the rarefaction plot.

e. Comparative Analysis

This includes calculation of beta diversity which estimates the differences in species diversity  between samples which is represented through a PCoA plot.

Deliverables :

• Quality filtration of data,
• QIIME/QIIME2 Analysis
• Pie charts for each taxonomic class.
• Abundance and identification of microbial community,
• Taxa identification
• Rarefaction curve and alpha diversity,
• Comparative analysis
• Heat map
• Beta diversity (PCoA Plot) (if more than 3 samples),
• Krona plot
• NCBI submission (SRA)
• Phylogenic analysis
• Functional Analysis of 16S using Picrust
• KEGG pathway distribution
• COG distribution
• Comprehensive report with publication standard methodology, graphs and tables.