MICROBIOME

AGRAJA RAJESH TIPARE

14BBT0010
Guide: Prof. Reena S. Rajkumari
WHAT IS MICRO
BIOME
➤ Microbiome is full collection of
genes of all the microbes in a
community. The Human
Microbiome can be considered as
counter part of human genome.
➤ Genes of microbiome outnumber
the genes in our genome by about
100 to 1.
HUMAN MICROBIOTA
➤ Human microbiota consist of 10-100 Trillion symbiotic microbial
cells harbored by each person, primarily bacteria in the gut; the
human microbiome consists of the genes these cells harbor.
➤ Gene catalog of 3.3 million non-redundant genes is reported in the
human gut microbiome alone, as compared to the ∼22,000 genes
present in the entire human genome.
➤ Individual humans are about 99.9% identical to one another in terms
of their host genome BUT 80-90% different from one another in
terms of the microbiome of their hand and gut.
MICROBIOME AND HEALTH
➤ Bacteria in the gut produce vitamins and break down our food; their
presence or absence has been linked to obesity, inflammatory bowel
disease and the toxic side effects of prescription drugs.
➤ Autoimmune diseases appear to be passed in families not by DNA
inheritance but by inheriting the family’s microbiome.
➤ Gut microbiome is different between obese and lean twins. Obesity
has also been associated with a poor combination of microbes in the
gut.
➤ Faecal microbiota transplantation (FMT) is a clinical procedure that
restores healthy bacteria in the colon
HUMAN MICROBIOME PROJECT [HMP]
➤ Worldwide research initiatives are mapping the human microbiome,
giving insight into uncharted species and genes.
➤ The purpose of the HMP is to study the human as a supra organism
composed of non-human and human cells, with the goal of
describing the human microbiome and analyzing its role in human
health and disease.
➤ HMP says human body has 100 trillion microscopic life forms living
on it.
➤ A person’s microbiome may influence their susceptibility to
infectious diseases and contribute to chronic illnesses.
 Human Genome
23000 genes
Human Microbiome
1,000,000+ genes
MICROBIOME WITH AGE
 AIM & OBJECTIVE
To compare and study the differences in the microbiome of
different individuals.
To find out the similarity and differences amongst the
microbiome of smokers, people eating paan to that of normal
individuals.
PROTOCOL
➤ Sample Collection
➤ DNA Extraction
➤ PCR [ 357F - 926RB ] v3 - v5 region amplification
➤ Sequencing [ 16s RNA ]
➤ Data Interpretation
SAMPLE
COLLEC
TION
SPUTUM SAMPLE
➤ Viscous fluid or suspension,
usually clear to milky. Infection is
often denoted by a yellow or
green colour.
➤ Preferably, the specimen should
be a first morning collection.
➤ If more than one specimen is
collected, they should be obtained
one per day on consecutive
mornings.
➤ Storage: 4-8 Degree
SPUTUM COLLECTION - PROCEDURE
➤ Gargle with water to rinse out your mouth.
➤ Take deep breaths through your mouth. Try and cough up mucous
from deep in your chest. You may need to take several deep breaths
and do lots of coughing.
➤ Open the jar and hold it close to your mouth. Spit the mucous into the
jar, without getting any on the outside of the jar.
➤ Screw the lid on tightly, so it does not leak.
➤ Put the used jar into the bag it came in and seal the bag closed.
➤ Put the specimen into the refrigerator until you can return the
specimens to the clinic or office. The specimen must be returned
within 3 days of collection.
 DNA
EXTRAC
TION
DNeasy PowerSoil Kit
DNA EXTRACTION
➤ Process of purification of DNA from sample using a combination of
physical and chemical methods.
➤ Essential components of DNA extraction includes: Maximize DNA
recovery, Inhibit nuclease, maximise quality of DNA.
➤ DNA should be free from protein, RNA and other cell components.
METHOD - DNEASY POWERSOIL KIT
➤ Fast and easy protocol isolates high-quality DNA from up to 250 mg
samples in just 30 minutes

Inhibitor Removal Technology removes PCR inhibitors found in tough sample

types

Isolate DNA with the DNeasy PowerSoil Kit. IRT eliminates humic acid content
leaving highly pure DNA for downstream applications analysis. PCR analysis can
detect DNA from a variety of organisms including fungi, algae, Gram (+/-) bacteria
(e.g., Bacillus subtilis, Bacillus anthracis), actinomycetes (e.g., Streptomyces) and
nematodes. 
16S RNA SEQUENCING
➤ 16S ribosomal RNA (rRNA) sequencing is a common amplicon
sequencing method used to identify and compare bacteria present
within a given sample.
➤ NGS-based 16S rRNA sequencing is a cost-effective technique to
identify strains
ANALYS
IS OF
MICROB
IOME
Tools & Techniques
ANALYSIS OF MICROBIOME
➤ There are several pipelines for analysis of microbial community data such
as mothur, WATERS, the RDP pyroseqeuncing tools, and QIIME.
➤ QIIME is a free, open-source platform for the analysis of high-
throughput sequencing data that enables users to import raw sequence
data and readily produce measures of inter- and intra-sample diversity. 
➤ UniFrac- A robust method for comparing the differences between
microbial communities, which measures the proportion of shared branch
lengths on a phylogenetic tree between samples.
➤ UniFrac coupled to PCoA, has the ability to distinguish differences
between communities utilizing as little as 10 sequences per sample
“
Thank You
REFERENCE
➤ Evaluation of Methods for the Extraction and Purification of DNA from
the Human Microbiome Sanqing Yuan1,4, Dora B. Cohen1,4, Jacques
Ravel5, Zaid Abdo2,3,4, Larry J. Forney1,4*
➤ Metagenomic study of the oral microbiota by Illumina high-throughput
sequencing Vladimir Lazarevic a,⁎,1, Katrine Whiteson a,⁎,1, Susan Huse
b, David Hernandez a, Laurent Farinelli c, Magne Østerås c, Jacques
Schrenzel a, Patrice François a
➤ 16S 454 Sequencing Protocol HMP Consortium Authors: Jumpstart
Consortium Human Microbiome Project Data Generation Working Group
➤ Qiime Community Profiling, Antonio González, Jose C. Clemente, Dan
Knights, Rob Knight