Next Generation Sequencing Platforms –Sequencing by synthesis (SBS) 454/Pyrosequencing...

Next Generation Sequencing Platforms

– Sequencing by synthesis (SBS)• 454/Pyrosequencing• Illumina/Solexa• Helicos • Pacbio• (Charge-based detection system, Now-sequencing)

– Sequencing by hybridization• SOLiD

Pyrosequencing: Sequencing-By-Synthesis

CSB2008 August 2008 UCSC Sequencing Center

454 Sequencing SystemChemistry & PlatformSequencing-by-synthesisLibrary PreparationEmulsion Based Clonal Amplification

SOLiD BioanalyzerChemistry & PlatformLibrary ConstructionEmulsion PCR

ApplicationsFragment SequencingTranscriptome StudiesPaired-End SequencingTargeted sequencingSmall RNA


1) Prepare Adapter Ligated ssDNA Library (A-[insert]-B) 2) EmPCR: Clonal Amplification on 28 µ beads followed by enrichment

4) Perform sequencing-by-synthesison the 454 Sequencer

3) Load beads and enzymes in PicoTiter Plate™

Overview of The 454 Sequencing System


Mix DNA Library & capture beads(limited dilution)

Emulsion Based Clonal Amplification

“Break micro-reactors”Isolate DNA containing beads

• Generation of millions of clonally amplified sequencing templates on each bead• No cloning and colony picking

Create “Water-in-oil”

emulsion

+ PCR Reagents

+ Emulsion Oil

Perform emulsion PCR

Adapter carrying library DNA

A

BMicro-reactors


Centrifuge Step

Load Enzyme Beads

44 μm

Load beads into PicoTiter™Plate

Depositing DNA Beads into the PicoTiter™Plate


Sequencing By Synthesis

DNA Capture BeadContaining Millions of

Copies of a Single Clonal Fragment

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

Anneal Primer

Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells

Pyrophosphate signal generation

T

PPi

ATP

Light + oxy luciferin

Sulfurylase

Luciferase

APS

luciferin

Sequencing-By-Synthesis


Sequencing Workflow Overview

Clonal amplification of fragments bound to beads in microreactors

One Bead

One Read 400,000 reads per run

One Fragment

Generation of small DNA fragments via nebulization

Ligation of A/B-Adaptors flanking single-stranded DNA fragments

Emulsification of beads and fragments in water-in-oil microreactors

Sequencing and base calling

Sample input: Genomic DNA, BACs, amplicons, cDNA


Sequencing Workflow Library Preparation

8.0 h

7.5 h

4.5 h and 10.5 h

DNA library preparation and titration

emPCR Sequencing

sstDNA library created with adaptorsA/B fragments selected using streptavidin-

biotin purification

Genome fragmented by nebulization


Sequencing Workflow Emulsion PCR

8.0 h

7.5 h

4.5 h and 10.5 h


emPCR Sequencing

Emulsion-based clonal amplification

Anneal sstDNA to an excess of DNA Capture Beads

Emulsify beads and PCR reagents in water-in-oil microreactors

Break microreactors, enrich for DNA-positive beads

Clonal amplification occurs inside microreactors


Sequencing WorkflowLoading of PicoTiterPlate

Device

• Well diameter: average of 44 µm• > 400,000 reads obtained in parallel• A single clonally amplified sstDNA

bead is deposited per well

Depositing DNA beads into the PicoTiterPlate device

Quality filtered bases

Amplified sstDNA library beads


Sequencing WorkflowSequencing by Synthesis

• Bases (TACG) are flowed sequentially and always in the same order (100 times for a large GS FLX run) across the PicoTiterPlate device during a sequencing run.

• A nucleotide complementary to the template strand generates a light signal.

• The light signal is recorded by the CCD camera.

• The signal strength is proportional to the number of nucleotides incorporated.

8.0 h

7.5 h

4.5 h and 10.5 h


emPCR Sequencing

FlowgramKey sequence


GS FLX Data AnalysisFlowgram Generation

Flowgram

Key sequence = TCAG for signal calibration

Flow Order

1-mer

2-mer

3-mer

4-mer

TACG

TTCTGCGAA


GS FLX Data AnalysisOverview

GS De Novo Assembler

GS Reference Mapper

GS Amplicon Variant Analyzer

Image capture

Image processing

Signal processing GS Run Browser

T

AG

CT

T

AG

CT


GS FLX System PerformanceRead Length


The Genome is comprised of repeat regions

• Depending upon the specific genome characteristics, microreads (~25 bp’s) cover only a portion of the genome– In human – 25 base pair reads can only be mapped

uniquely to 80% of the genome

• Short reads are limiting in known genomes – What about unknown genomes?

– Mapping versus de novo assemblies– Mapping will miss genome rearrangements– Mapping is only as good as the reference


Why Does Length Matter?

• Longer sequencing reads mean more applications

– Identify and characterize small and short RNA’s– Full length cDNA sequencing for expression levels and variations– Amplicon resequencing for genetic variation including somatic mutations– Sequencing of micro-organisms in a single instrument run– Sequencing of complex genomes – mammalian & plant– Sequencing of complex samples – Metagenomics, Ancient DNA


Longer sequencing reads mean more applications

– HIV Studies (3) – ChIP-Sequencing (8)

• Boyle et al, Cell: Mixed technologies for mapping open chromatin– Metagenomics (12)

• Palacios et al, New England Journal of Medicine, Pathogenic Virus Detection

– Whole Genome Sequencing (30) • Velasco et al, PLoS: Pinot Noir Genome

– Paired-End sequencing• Detecting Structural Variations across two human genomes

– Technology and Bioinformatics (11)• Meyer et al, NAR,: Using Picogram quantities of sample

– Transcriptome studies – cDNA (17)– Small RNA (32)– Amplicon and Methylation Studies (9)


Applications of Whole Genome, Ultra Broad and Ultra Deep HT- Sequencing

HT- Sequencing Technology Applications

Whole Genome Sequencing

VirusBacteriaFungusHigher EukaryotesHuman

Ultra DeepSequencing

Population Biology

HIV

Bacterial 16S

ResistanceTropism

Amplicons

Ultra Broad Sequencing

Small RNASAGE/CAGE

Metagenomics

Expression

Novel strain ID

Transcriptome

HLA Typing


The power of Metagenomics

• How to Identify an environment based upon the microbial organisms that are present– Microbial Population Structures in the Deep Marine Biosphere

• Huber et al., Science, 318, p97, 2007

• Determining the state of an environment based upon the presence and mixture of microbial organisms

– The interdependence of Coral and it’s microbial environment• Wegley et al., Environmental Microbiology, 9, p2707, 2007

• Detecting viral pathogens – quickly and accurately – Less than 12 months from first identification of affected hives to possible

pathogen• Cox-Foster et al., Science, 2007

– Transplant victims from Australia• Palacios et al, New England Journal of Medicine, 2008


Transcriptome AnalysisWorkflow Comparison

GS FLX (clonal sequencing ensured through emPCR)

Sanger (E. coli cloning, often concatemerization)

cDNA libraries(short tag library, EST library)

Concatemerization,insert fragments into vectors and clone into bacteria

Grow, pick colonies

Template Generation

Sequencing

Time: Weeks

emPCR

Sequencing Time: Days

cDNA libraries(short tag library, EST library)


• Sequencing of approximately 400,000 small RNAs from C. elegans

• Another 18 unknown miRNA genes were detected

• Thousands of endogenous siRNAs acting preferentially on transcripts associated with spermatogenesis and transposons were identified

• A new class of small RNAs was identified: 21U-RNAs. They all begin with an U and are precisely 21 nt long.


Multiplex Identifier Basics• What is it?

– Two new kits, each with 6 different library adapters (total of 12 adapters)– Each MID library adapter has an added, specially encoded 10-base region– Used to “bar-code” up to 12 different genomic library samples to be run in

the same region of a single sequencing run

Primer A MID 1Key Library fragment Primer B

Seq. primer Read

#bases: 15 4 10

Primer A Key Library fragment Primer B#bases: 40 4

MID Library

Standard Library Seq. primer Read

Primer A MID 2Key Library fragment Primer B

Primer A MID nKey Library fragment Primer B


Paired-End Applications

• ~100 bp sequencing tags separated by 3 kb spacing• Use for de novo assembly

– Order contigs• Use for Structural Variation studies

– Inversions, Deletions, Insertions…– High resolution detection – 3kb spacing vs 10 to 40 kb


Paired-Ends workflow


Targeted Enrichment of Human gDNA

gDNAExon 1 Exon 2 Exon 3 Exon 4 Exon 5

Fragment and hybridize to NimbleGen capture array

HT-SequencingAnalyze

Exon Sequences

Elute


Sequencing all the known exons from the human genome

• “Direct selection of human genomic loci by microarray hybridization,” – Albert et al., Nature Methods, (4) 11, 903 -905, 2007

• ~6,700 gDNA loci selected• BRCA1 region

2 MB Region


Another Sequence-Capture Example

• 19 Kb region from Chromosome 4

GS FLX Seq Reads

Sequencing Coverage

Seq-Cap Array Probes

Targeted Exons


SOLiD Library Preparation

The SOLiD system uses either a fragment library or a mate-paired library depending on the user’s desired information or application.


Emulsion PCR and Bead Enrichment

PCR takes place in oil in water microreactors. Post-PCR, templated beads are separated from non-templated beads, and modified at the 3’ end to allow covalent

linkage to the SOLiD sequencing slide.


Bead Deposition

Beads are deposited into 1,2,4, or 8 segmented chambers on a slide.


Sequencing By Ligation and Data Analysis

Primers hybridize to adaptors and a set of 4 dye labeled probes competes for ligation to the primer with probe specificity determined by the 4th and 5th base interrogation

during each ligation series, for 5-7 rounds.

After each round of ligation, a new primer offset by 1 base is hybridized for a new round of ligations. 25-35bp are generated through 5 sequential primer reset and

ligation rounds.


Library Construction

2 different libraries can depending on the application and desired information.


Fragment Library

DNA is fragmented and PCR primer adaptors are ligated to the DNA


Mate-Pair Library

DNA is sheared, selected for a desired input size, and circularized around an internal adaptor.


Mate-Pair Library (cont.)

The circularized DNA is enzymatically cleaved to yield 2 DNA fragments separated by an internal adaptor. PCR primer adaptors are ligated on to the end of this piece of DNA.


Emulsion PCR (ePCR)

PCR takes place in oil in water microreactors containing P1-coupled beads, templates,

primers, and all required PCR reaction components..


ePCR


Emulsion PCR yields both monoclonal (unique templates and polyclonal beads (multiple templates), as well as some non-templated beads.


Clonal Amplification


Post-Emulsion and ePCR


Bead Enrichment

Templated beads are separated from non-templated beads via polystyrene beads


Pre- and Post-Bead Enrichment P2-hybridization


Bead Deposition

Templated beads are modified at their 3’-end and covalently attached to a glass slide.


Slide Configurations


SOLiD Sequencing Chemistry


4-color Ligation Reaction


A complementary dye-labeled probe hybridizes is ligated to the universal sequencing primer.


De-phosphorylation

A phosphatase cleaves the 3’-phosphate from the universal sequencing primer preventing ligation to templates where no probe was ligated.


Visualization

The fluorescently labeled dyes attached to each probe are visualized.


Cleavage

Fluorescent dye labeled nucleotides are cleaved from hybridized probe.


2nd Ligation Cycle

The next set of labeled probes hybridize and are ligated to the previously hybridized probe.


2nd Cycle Visualization


2nd cycle cleavage


Every 5th base is interrogated


Reset

Template is stripped


1st cycle after reset

A new set of universal sequencing primers is hybridized, offset by (n-1).


1st cycle after reset


2nd round


Sequential Rounds of Sequencing with Fragment Library

In each round of sequencing ………….??????????????


Sequential Rounds of Sequencing with Mate-Paired Library

In each round of sequencing ………….??????????????


Di-Base Encoding


Advantages of di-Base Encoding


Advantages of di-Base EncodingReal SNPs


Advantages of di-Base EncodingMiscall


Only Certain Transitions are Allowed for Real SNPs


Only Allowed Transitions


Two color changes not allowed


Benefits or solid


Thank you for your attention

Next Generation Sequencing Platforms –Sequencing by synthesis (SBS) 454/Pyrosequencing...

Documents

Transcript of Next Generation Sequencing Platforms –Sequencing by synthesis (SBS) 454/Pyrosequencing...