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Metagenomic Sequencing for V3-V4 Region

Metagenomics is the study of the functional genomes of microbial communities while 16S sequencing offers a phylogenetic survey on the diversity of a single ribosomal gene, 16S rRNA. The marker allows one to examine genetic diversity in microbial communities, specifically to find out what microbes are present in a sample.

The main reason for using V3/V4 region in majority of the cases is that these regions contain the maximum nucleotide heterogeneity and displays the maximum discriminatory power. However, it should be noted that no single region can differentiate for all bacteria.

Because of the complexity of DNA–DNA hybridization, 16S rRNA gene sequencing is used as a tool to identify bacteria at the species level and assist with differentiating between closely related bacterial species. Many clinical laboratories rely on this method to identify unknown pathogenic strains.

BRIEF DESCRIPTION & SCOPE OF WORK:

The Following Steps are involved in V3-V4 Region Sequencing –

• Sample QC
• Library Preparation
• Sequencing
• Bioinformatics Analysis

 

Sample QC:

• Sample QC will be carried out by agarose gel electrophoresis, nano drop Quantification will be done by using qubit

Library Preparation:

• Library will be prepared separately using 16s specific primers for all samples
• Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent Tape station kit.

Sequencing:

• Prepared library will be sequenced using 2 x 300 bp paired end sequencing using latest sequencing chemistry on Illumina MiSeq platform.
• Delivery of data up to ~100000 paired end reads per library.

Bioinformatics Analysis:

• Quality filtration of data
• QIIME Analysis
• Pie charts for each taxonomic class
• Heat map
• abundance and identification of microbial community
• Taxa identification
• Rarefaction curve and alpha diversity
• Comparative analysis

Sample Requirement:

• 1 ug of Genomic DNA with RNase treatment, 260/280 value not less than 1.8-2.00