16S/18S/ITS microbiome profiling

16S ribosomal RNA (rRNA), 18S/ITS sequencing is a widely accepted targeted sequencing method used to identify and compare bacterial and fungal community/population present within a particular sample. Targeted metagenomic sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiome or environments that are difficult or impossible to study. Microbiome sequencing approach relies on sequencing of 16S ribosomal RNA (rRNA), ITS gene as the genetic marker to study bacterial phylogeny and taxonomy. This genetic marker contains conserved hypervariable regions (V3 & V4) for bacteria and ITS regions for fungi, which can be used for identification, harnessing the power of PCR to generate amplicon are sequenced with NGS (pair end sequencing of 2X300bp chemistry) to explore phylogenetic classifications such as genus or species in diverse microbial populations. Each sample require to generate 0.1-0.2 million read of each of samples for comparative analysis. Thanks to multiplexing approaches, several amplicons from several samples can be analyzed in parallel to reduce costs per sample.

Eurofin offers comprehensive end to end solution of study of your 16S rRNA and fungal microbiome profiling including Bioinformatic analysis which will help to align your microbiome study.

Methodology

Sample Requirement:

• Submit 1-3 µg of RNA-free genomic DNA (NGS grade) of high molecular weight, NanoDrop A_260/280 ratio >1.8; A_260/230=2.0-2.2; and at a concentration of ~50 ng/µl
• Or soil, plant/ human microbiome, water or sludge samples shipped in cool packs to Eurofins facility.

Sample QC:

• RNA free NGS grade gDNA isolation will be carried out using commercially available and well validated DNA isolation kit.
• Quality checking will be carried out using agarose electrophoresis and NanoDrop. Quantification will be carried out using Qubit 3.0 Fluorometer.

Library Preparation:

• Amplicon libraries will be prepared using 16S rDNA gene specific primers and indexing using Nextera XT Index Kit.
• Library validation will be carried out using Agilent 4200 Tape station kit.

Sequencing:

Sequencing will be carried out on Illumina MiSeq platform using 2 x 300 bp paired end sequencing chemistry.

Deliverable

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services 

Turnaround time

5-7 weeks after arrival of your samples and all necessary information. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples.