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Next Generation Sequencing Presentation

This presentation is the summary of the methods of Next generation sequencing. This also includes the applications that have been recently published

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1K views28 pages

Next Generation Sequencing Presentation

This presentation is the summary of the methods of Next generation sequencing. This also includes the applications that have been recently published

Uploaded by

kalyankpy
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT or read online on Scribd
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Emerging Science:

The Next-Generation Sequencing (NGS) Technology

Kalyan Kumar Pasumarthy


The Power of Next-Generation Sequencing
Sanger’s di-deoxy method vs NGS

Its cheaper
• $2700 million vs $1.5 million

Its faster
• 13 years vs 5 months
Various applications
• whole genome sequencing
• targeted genomic resequencing
• metagenomics
• transcriptomics
• chromatin analysis
• DNA-protein binding analysis
Next-Generation Sequencing Platforms
A Glimpse of Next-Generation Sequencing

Vendor Roche/454 Illumina/Solexa ABI/SOLiD Helicos


Sequencing by Sequencing by True single molecule
Technology Pyrosequencing
synthesis ligation synthesis

Platform Ti IIx 3 Heliscope

Reads (in million) 1.25 250 320 600

Read length (bp) 400 100 50 35

Run time* (days) 0.4 5 8 8

Yield (in Gigabase) 0.5 25 16 21-35


Rate (in 1.25 5 2 1/hr
Gigabase/day)
* Depends on the experiment
Data Processing

Base calling
Roche/454 – GS FLX
• Clonal amplification by emulsion PCR

• Bead deposition on picotitre plates


Roche/454 – GS FLX
• Pyrosequencing technology

A T G C A T
Illumina/Solexa – Genome Analyzer
• Clonal amplification by bridge PCR isothermally
Sequencing by reversible dye-terminators
ABi- SOLiD (Supported Oligonucleotide Ligation and Detection)

• Clonal amplification by emulsion PCR

• Sequencing by ligation

• Template is probed by fluorescent tagged


di-nucleotide probe
Primer n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Primer n-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Primer n-2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Primer n-3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Primer n-4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Helicos - Heliscope
• No amplification

• tSMS technology – True Single Molecular Synthesis

A T A T A T T T T
A A C A A C
C C C C C C
G G A G G A
A G T A G T
T C G A T C G
T A TA TA T A T A T A
T A TA TA T A T A T A
T A TA TA T A T A T A
T A TA TA T A T A T A
AAAAA T T T T T T
T T T T T T

Template bound to Addition of a fluorescent


Template preparation adapters on the flowcell labelled base
T T T G T G T G T T T T
A A C A A C A A C
C C C C C C C C C
G T A G T A G T A
G G T G G T G G T
A T C G A T C G A T G C G
T A T A T A T A TA TA T A T A T A
T A T A T A T A TA TA T A T A T A
T A T A T A T A TA TA T A T A T A
T A T A T A T A TA TA T A T A T A
T T T T T T T T T
T T T T T T T T T

Fluorescent label Probe with another Polymerase adds


removed labelled base the labelled base

C C C
T T T T T T T T T
A A C A A C A A C
C C C C C C C C C
G T A G T A G T A
G G T G G T C G C G T
A T G C G A T G C G A T G C C G
T A T A T A T A T A T A T A T A T A
T A T A T A T A T A T A T A T A T A
T A T A T A T A T A T A T A T A T A
T A T A T A T A T A T A T A T A T A
T T T T T T T T T
T T T T T T T T T

Fluorescent label Probe with another Polymerase adds


removed labelled base the labelled base
Pacific Biosciences – Single Molecule Real Time Technology

• SMRT technology

• Sequencing done in SMRT cells Zero-mode wave guides

• Exploits the RCR mode of replication by Φ29 DNA polymerase

43.5 µm X 32.8 µm ZMW Detection effeciency


Pacific Biosciences – Single Molecule Real Time Technology

Bases are individually labelled at γP


Various applications

• Whole genome sequencing

• Targeted genomic resequencing

• Metagenomics

• Transcriptomics

• Chromatin analysis

• DNA-protein binding analysis


Whole Genome Sequencing

• Best method at present is Roche/454 GS FLX

• Long reads
• Mycoplasma genetalium 96% coverage and 99.9% accuracy (Marguiles, 2005)
• James Watson at 7.4 fold redundancy(Wheeler, 2008)
• AML complete genome sequecing identified 8 non-synonymous mutations in
comparision to normal skin genome of effecter person (Ley, 2008)

• Helps in mapping genomic structural variation : insertions,


deletions & rearrangements

•1000 genome project aimed at characterising and cataloging of


human genetic variation
Targeted Genomic Resequencing

• To identify the genetic variation in genomic subregions

• Helps in identifying polymorphisms and mutations in genes


implicated in cancer and in regions where whole-genome association
studies have implicated in disease

• SNPs were catalogued in a region of 134 kb from 79 people (Yeager, 2008)

• 1000 mutations were identified in 23 genes by sequencing 623 genes from


180 cancer samples (Ding, 2008)
Metagenomics

• Study of genetic material recovered directly from environmental


samples

• Microbial diversity from environmental and clinical samples can be


studied

• Marine Biodiversity (Huber 2007)

• Soil Biodiversity (Urich, 2008)

• Oral cavity: 22 phyla with 19,000 phylotypes (Keijser, 2008)

• Gut metagenome from lean and obese twins identified that obesity is
associated with phylum level changes in microbiota, reduced bacterial
diversity and altered representation of bacterial genes and metabolic
pathways (Turnbaugh, 2009)
Transcriptomics

• RNA analysis by sequencing: RNA-seq

• Qualitative and quantitative analysis is not limited by the


knowledge of genome

• Helps in distinguishing RNA isoforms, allelic expression, alternative


splicing events, demarcation of exon-intron boundaries

• Identification of transcripts from unidentified genes/pseudo genes

• Overlapping genes were identified in yeast (Nagalakshmi, 2008)

• Small RNAprofiling in Arabidopsis (Lister, 2008), Maize (Nobuta, 2008),


Tomato (Moxon, 2008) Medicago ( Szittya, 2008), Rice under abiotic
stress conditions (Zhou, 2009)
Mapping of DNA-Protein interactions

• Chromatin immuno-precipitation is followed by sequencing

• Advantageous over ChIP-chip

• Not limited by the genome knowledge

• Human neuro restrictive silencing factor (NRSF) binding sites identified


(Johnson, 2007)

• STAT1 (Robertson, 2007)

• Histone modification and Chromatin accessibility can be assayed at genome


level
• H2A.Z histone genome wide positioning in yeast (Albert, 2007)

• Chromatin accessibility in C. elegans (Johnson, 2006)


How does this Technique help us?

• In addressing the transcriptome (ESTs) during stress conditions


• small RNA profiling under various conditions
• Abiotic stress
• Biotic stress
• Tomato
• Rice
• Cotton

•To examine the chromatin status in varying physiological conditions

• Analysis of the microbiota of the root and soil

• Analysis of the transcriptional activation of various genes by DNA


binding proteins

• viral proteins that bind DNA and show transcriptional activation


• genes modulated by various stress responsive transcription factors
Limitations

• Cost

• Error rate (Higher than Sanger sequencing ~ 1%)

• Short-read length

• Lack of effective computational algorithms

• Expertise in India is very meagre

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