Rna sequencing and its method

2. j
RNA Sequencing and
Methods
Shama Iftikhar

3. Content……
RNA
SEQUENCING
RNA sequencing
Ways for RNA sequencing
Why we need RNA
sequencing
Methods
RNA isolation
Technology used for isolation
RNA selection / depletion

4. Content……
Cdna synthesis
Sequencing
ILLUMINA
sequencing
Tagmentation
Amplification
Sequencing

5. RNA
RNA stands for ribonucleic
acid.
It is single stranded nucleic
acid.
 RNA contain Uracil instead of
Thymin.
RNA is vital for living beings.
Genetic information is transfer
in the form of RNA

6. Sequencing
The process of
determining the
correct order of amino
acids in a polypeptide
chain of a protien, of
nucleotide with in a
DNA and RNA.

7. RNA sequencing
The whole transcriptom
shotgun sequencing uses
Next Generation
sequencing to reveal the
presence and quantity of
nucleotide in RNA sample.

8. Ways of RNA
sequencing
There are two ways to for RNA sequencing
Direct method of RNA sequencing
The RNA sequencing in which the RNA is
directly sequenced without changing into the
double stranded Cdna is called Direct
method.
Indirect method of RNA sequencing
The RNA sequencing in which the RNA is
converted into the double stranded C dna is
callled Indirect method of sequencing.

10. Why we need
sequencing
RNA sequencing helps to
look at
Alternative gene spliced
Transcripts
Post transcriptional
modification
Gene fusion
Mutation
Change in Gene
expression
RNA structure

11. Why we need
sequencing

12. Why we need
sequencing

14. Methods
Library preparation includes
following steps;
 RNA isolation
 RNA selection/ depletion
 cDna synthesis
 Sequencing

15. RNA Isolation
RNA is isolated from tissues with
special techniques and due to its
fragile structure handle with special
care.
R-nase group enzyme that demage
the RNA structure , So R-nase free
solution is used;
Chloroform
Alcohol

16. Techniques use for
isolation
Following techniques uses for RNA
isolation;
 Magnetic – bead Technology
Silica Technology
Lithium Chloride and Urea isolation
Capillary electrophrosis
Glass fiber filters

17. Quality measurement
Agilent Bioanalyzer
 It produces an RNA Integrity Number
(RIN) between 1 and 10 .
 10 is the highest quality samples.
The RIN estimates sample integrity
using gel electrophoresis and analysis of
the ratios of 28S to 18S ribosomal bands.

18. Quality measurement
Low-quality RNA (RIN
< 6) can affect the
sequencing result and lead
to erroneous biological
conclusions.

20. RNA selection
RNA is filtered with
3poly A tail to include
only mRNA .
rRNA depletion is
performed by mixing the
Poly A with Poly T
Oligomers, covalently
attached to substrate
typically magnetic beads.

21. RNA may in the form of TOTAL RNA .
It includes the The total RNA pool includes
ribosomal RNA (rRNA), precursor messenger
RNA (pre-mRNA), mRNA, and various classes
of noncoding RNA (ncRNA). Depletion of
rRNA using commercially available kits, such
as RiboMinus (Life Technologies) or RiboZero
(Epicentre).
Continue…

23. DNA sequencing technology is
more mature, so the RNA is reverse
transcribed to cDNA
 Fragmentation and size selection
are used for the selection of RNA
with correct size and shape for the
sequencing machine.
Fragmentation and size selection is
done by action of enzymes.
Cdna synthasis

27. The actual RNA sequencing
varies significantly depending
on the platform used.
 Pyrosequencing on the 454
Life Sciences platform.
polymerase-based sequence-
by-synthesis on the Illumina
platform.
Sequencing by ligation on
the ABI Solid
Sequencing platform.
Sequencing

30. It was invented by Bruno Canard and
Simon Sarfati at the Pasteur Institute in
Paris.It was developed by Shanker
Balasubramanian and David
Klenerman of Cambridge University.
Procdure
RNA is purified,selected and fragmented.
Tagmentation
Amplification
sequencing
ILLUMINA sequencing

31. Tagmentation
Enzymes called
transposases randomly
cut the DNA into short
segments ("tags").
 Adapters are added on
either side of the cut
points (ligation).
Strands that fail to
have adapters ligated are
washed away.

32. Clusters are generated by bridg
amplification Polymerases
move along a strand of DNA,
creating its cdna.
 The original strand is washed
away, leaving only the reverse
strand.
 At the top of the reverse strand
there is an adapter sequence.
Bridge amplification

33. .
The DNA strand bends and attaches to
the oligo Polymerases attach to the reverse
strand, and its complementary strand is
made.
Now double stranded DNA is denatured
so that each strand can separately attach to
an oligonucleotide sequenc. One will be
the reverse strand; the other, the forward.
This process is called bridge
amplification.
Bridge amplification

37. Primers and modified nucleotides enter the chip.
These nucleotides have reversible 3' blockers
that force the polymerase to add on only one
nucleotide at a time as well as fluorescent tags.
 After each round of synthesis, a camera takes a
picture of the chip.
 A computer determines what base was added by
the wavelength of the fluorescent tag and records
it for every spot on the chip.
Sequencing

38. Sequencing
After each round, non-incorporated
molecules are washed away.
The process continues until the full DNA
molecule is sequenced.

42. Abecasis GR, Cherny SS, Cookson WO,
Cardon LR. Merlin—Rapid analysis of dense
genetic maps using sparse gene flow trees.
Adams MD, Kelley JM, Gocayne JD, Dubnick
M, Polymeropoulos MH, Xiao H, Merril CR, Wu
A, Olde B, Moreno RF, et al. Complementary
DNA sequencing: Expressed sequence tags
and human genome project. Science.
Denoeud, F. et al. Annotating genomes with
massive-scale RNA sequencing. Genome
Biol. 9, R175 (2008)
www.illumina.com.
Refrence