Next Generation Sequencing: An Overview
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Next Generation Sequencing:
An Overview
DNA Sequencing • Refers to determining the order of nucleotide (G, A, T, and C) in a stretch of DNA • Useful in biotechnology research and discovery, diagnostics, and forensics.
What is Sequencing?...............How does it work? DNA Sequencing DNA sequencing = determining the nucleotide sequence (A, T, G, and C) of the DNA of a gene. We are currently using the Sanger’s method.
Sanger Sequencing • Utilizes dideoxunucleotides triphosphates to terminate DNA chain elongation. • Separation of molecules by gel or capillary electorphoresis and detection of dye-labelled terminator • Can be used to interrogate the sequence of single samples. • 96 samples can be run in SBS format with 24-36 runs per day. A single instrument can generate 1-2 million bases per day.
Sanger Sequencing Workflow
Sanger Sequencing Pipeline
Library Picking &
Template Prep
Construction Growth
Plasmids
PCR
Amplicons
PCR Cycling Seq. Setup / AxyPrep Mag Plasmid
& CleanUp Cycling
AxyPrep Mag PCR Clean up
AxyPrep Mag PCR Normalizer
Ready to Sequence Templates
3730xl
Seq Setup/ 3730 XL
Analysis
Cycling Analysis
AxyPrep Mag DyeClean
From Slab Gels to Single Molecule Sequencers
• Late 1980s Slab Gel sequencers using radioactive isotopes
and later Fluorescence chemistry (10 Kb per 4 hr run).
• Late 1990s Capillary sequencers (50 Kb per 1hour run)
• 2005 Massive parallel pyrosequencing (20 MB per 5 hr run)
• 2007 Sequencing by synthesis (1 GB per 5 day run)
• 2010 Single molecule sequencing (100 GB per 5 day run)
• 2013 Human genome in 15 minutes
8
Next Gen Sequencing
• Employs micro- and nanotechnologies to reduce the size of sample
components, reducing reagent costs, and enabling massively parallel sequencing
reactions.
• Highly multiplexed, allowing simultaneous sequencing and analysis of millions of
samples.
Multiple cycles
T G C T A C
Sanger vs. Next Gen
Next Gen v. Sanger
Traditional Sequencing vs. Next Generation Sequencing: Data Throughput
1 x Illumina GAII 200+ of 3730xl
Vs.
Days vs. Years
The Sequencing Landscape is ChangingNext Generation Sequencing
PlatformsSecond Generation Sequencing Throughput
Illumina Genome Analyzer IIx Roche GS FLX Life Technologies SOLiD 3
Plus
• Sequencing by synthesis using • Sequencing by synthesis using
reversible flurorescent dye chemiluminescence detection; • Sequencing by ligation; in vitro
terminators using clonal single sample prep;
• 400 to 500 base reads
molecule array; • 35 to 2 by 50 base reads;
• 1 million fragments of DNA in
• 35 to 50 base reads length • 500 million to 1 billion shotgun
parallel on picotitre plate
• 138 to 336 million shotgun reads reads per 2 slide run;
• 400 MB per 10 hr run
per run; • reference sequence required
• 4.5 to 36 GB per 2 to 9.5 day run • 12 – 48 GB per 3.5 to 14 day 2-
slide run.Differentiating Next Gen technologies
Sheared Library Clonal Sequencing
construction template
DNA
amplification
Clonal amplification via Massively parallel sequencing-by-synthesis
Illumina Library Construction
bridge amplification of DNA clusters
Clonal amplification with Massively parallel pyrosequencing of bead
454 Library Construction
emulsionPCR and bound DNA templates
enrichment
Clonal amplification with Massively parallel ligation-based
SOLiD Library Construction
emulsionPCR and sequencing of bead bound DNA templates
enrichmentComparison of Next Gen Technologies
GS FLX Genome Analyzer SOLiD
Library Fragment, Mate- Fragment, Mate- Fragment, Mate-
Construction Paired Paired, Paired-End Paired
Sequencing Sequencing by Sequencing by Sequencing by
Chemistry Synthesis Synthesis Ligation
DNA Support 25-35 µm bead Flow cell surface 1 µ bead
Amplification Emulsion PCR Cluster Emulsion PCR
amplification
Sequencing High density well- 8-channel flow Single slide
Reaction Surface plate cell imaged in panelIllumina Genome Analyzer
(GA)Illumina Genome Analyzer
(GA, GAII, GAIIx)
• DNA Libraries bound to a 8 channel flowcell
• Sequencing by synthesis using reversible terminators cBot
• Detection of fluorescent tagged bases
• Readlengths of up to 2 by 100 bases.
Paired end module
Cluster stationIllumina workflow Library construction 1-4 days depending on application Cluster Amplification (Cluster station) Automated, approx 1 hour to set up, 5 hours run time Sequencing (GAII and Paired End module) Approx 2-9 days depending on read length and number of reads
Illumina GA - Cluster Generation Flow cell, reagents and samples loaded onto cluster station Aspirates samples and reagents into flow cell Automates the formation of amplified clonal clusters from single DNA molecules Approx 5 hours run time, 1 hour hands on time
Template amplification: no beads, no
emulsions
“Cluster generation”
(walk-away)Illumina GA - Cluster Generation
(cont)Illumina GA - Sequencing • Flowcell and reagents loaded onto Genome Analyzer. • 1-2 hour loading time • Walkaway automation • 2 - 9 days run time depending on read length
3’ 5’
Sequencing By Synthesis (SBS)
Cycle 1: Add sequencing reagents
First base incorporated
A
T
G Remove unincorporated bases
C C
G
T T Detect signal
A
C C Deblock and defluor
A G
T
A
PPP Base Fluor
G T
A
A
C
T C
C
G G
A Cycle 2-n: Add sequencing reagents and repeat
C T
C
G
A
T
5’Sequencing by Synthesis - 4
Fluors
Synthesis of 2nd strand Four nucs per cycleGenome Analyzer IIx - Specifications
Roche Genome Sequencer GS
FLXRoche GS FLX (GS20, FLX, Titanium) • Uses pyrosequencing – a process that uses chemiluminescence for detection. • Detection of light signal generated by luciferase when complimentary bases are incorporated into sequencing strand. • Libraries are bound to beads which are deposited onto a PPT plate for sequencing. • Long read lengths up to 400bp. • Run time approx 10 hours.
GS FLX Workflow
DNA Library Preparation emPCR and enrichment Sequencing
1 to 3 days 1.5 days 1 day
DNA Library Preparation emPCR Sequencing
Fragment DNA through nebulization Water-in-oil emulsion DNA beads
or other means. placed in pico-
Attach adapters to DNA fragments. Fixes adapter-ligated titre plate device.
Prepare single-stranded DNA library fragments to small Uses
with adapters. DNA-capture beads pyrosequencing
Recently, a rapid protocol has been chemistry
introduced which eliminates the Purification of Detection using a
need for making the adapter-ligated amplified DNA CCD camera
DNA fragments single stranded. colonies on beads 10 hr sequencing
runEmulsion PCR Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR
Enrichment Beads with amplified DNA are purified using magnetic enrichment beads. Approximately 1/3 of beads have a product.
GS FLX: Bead
deposition
DNA beads are loaded DNA beads packed
into the wells of the PTP. into wells with
surrounding beads
and sequencing
Empty PicoTiter slide enzymes.GS FLX: Instrument Loading
Genome is loaded Load PicoTiterPlate
into into instrument.
a PicoTiterPlate.
Load reagents in a Sequence entire genome
single rack. at once, in real-time.GS FLX: Sequencing-by-
synthesis
• Simultaneous
sequencing of million of
DNA library molecules in
a pico-titre plate..
• Pyrophosphate signal
generation upon
complimentary nucleotide
incorporation — dark
otherwise.
DNA capture bead
containing millions of
copies of a single
clonal fragmentGS FLX Sequencing-by-
synthesis
Repeated dNTP flow
sequence:
G T C A
Process continues until user-
defined number of nucleotide
flow cycles are completed.
A A T C G G C A T G C T A A A A G T C A
T T A G C C G T A C G C
A T T T T C
G
A T C
G T C
A G
A G
T
Anneal PrimerAdvantages/Disadvantages
• Advantages • Disadvantages
– Q20 read lengths of 400 – Difficulty getting through
bases (99% accuracy at the
homopolymers
400th base and higher for
preceding bases) – Each run is expensive and
– significantly higher hence not ideal for re-
throughput compared to sequencing applications
Sanger sequencing compared to the Genome
– Does not rely on cloning Analyzer and/or SOLiD
efficiency
– DNA libraries can be
barcoded and separated
during data analysis.Life Technologies SOLiD
Life Technologies SOLiD • Sequencing by Oligo Ligation and Detection • Libraries are bound to beads which are covalently attached to a glass support surface after emulsion PCR • Uses fluorescently labelled oligomers • Dibase encoding • Read lengths up to 2 by 50bp •Up to 8 samples/slide
Emulsion PCR
Enrichment P2’
Large
P1 Polystyrene P2
P2 P1
bead coated
with P2
Centrifuge in Supernatant
glycerol gradient Captured beads with templates
Pellet
Beads with no templateBead Deposition
3’-end
modification
Beads attached to glass
surface in a random arraySlide deposition and installation
Sequencing by Ligation
Sequence Data Analysis
• 4 dyes to encode 16, 2-base
combinations
• Each base is interrogated
by two probes, two
different ligation reactions
• Dual interrogation eases
discrimination errors
– Random or systematic vs.
True polymorphisms (SNPs)
Data is best analyzed in color space
- Leverages di-based advantagesSOLiD 3 Plus Specifications
Multiplexing
What is multiplexing? • Multiplexing: a method to analyze multiple biological samples in a single sample. • Barcodes are unique sequence identifiers added to samples during library construction. • Once barcodes are added, multiple libraries can be pooled together for emulsion PCR/cluster generation and sequencing. • Sequence data is then analyzed and traced back to each source.
Multiplexing
• Simpler workflow, ease-of-use
• Lower running costs
• Higher number of samples per run
Standard Protocol Multiplexing Protocol
8 Samples 16 Samples 128 Samples
8 Libraries 16 Libraries 128 Libraries
8 Emulsions 1 Emulsion 8 EmulsionsWhy it Multiplexing Important? Next generation DNA sequencing generates massive amounts of sequence data. Currently more data is generated per library than is required. To overcome this, researchers multiplex multiple libraries into single lane. However, generation of libraries is a bottleneck. Most researchers are not able to do this. SPRIworks will relieve this bottleneck by enabling researchers to make more DNA libraries faster.
Summary of Next Generation Sequencing Platforms
Examples of Next Generation Sequencing Applications De novo sequencing “De novo sequencing is the initial sequencing that results in the primary genetic sequence of organisms. A detailed genetic analysis of an organism is possible only after de novo sequencing has been performed.” - Applied Biosystems website Re-sequencing- looks for variation between strains or individuals cDNA Sequencing (Fragmented cDNAs)- Sequencing of transcribed regions Amplicon/PCR sequencing- could be targeted re-sequencing or possibly genome sequencing (i.e. viral).
You can also read
