overview of next generation sequencing platform … 2012/0305 - timmermann.pdf · next generation...

Next Generation Sequencing Core FacilityNext Generation Sequencing Core FacilityMax Planck Institute for Molecular GeneticsMax Planck Institute for Molecular GeneticsBerlin, GermanyBerlin, Germany

Dr. Bernd Timmermann

Overview of Next Generation Sequencing platform technologies

May 23rd 2012, Budapest

1. Technologies

Illumina

Roche / 454

2. Projects and Applications

whole Genome Re-sequencing

Sequence Capture

Amplicon Sequencing

3. Outlook

Outline


Max Planck Society

Max Planck Institutefor molecular Genetics

80 institutes and research facilities

20,435 people

Budget 1,400 million euro in 2010


1980 1985 1990 1995 2000 2005 2010

1000,000,000

100,000,000

10,000,000

1,000,000

100,0000

10,000

1000

100

10

Gel-based Systems

Capillary Sequencing

Next GenerationSequencing

First GenerationCapillary Sequencer

Second GenerationCapillary Sequencer

MicrowellPyrosequencing

Short-ReadSequencer

Thro

ughp

ut p

er s

yste

m [k

iloba

ses/

day]

Year

Modified after MR Stratton et al. Nature

458, 719‐724 (2009)

Development of Sequencing Throughput


Development of Sequencing Technologies

• 96 sequences in parallel• 3.2 billions of sequences

per run

Human Genome Project1000 Genomes Project


7 x Illumina

3 x SOLiD

5 x Roche GS

Sequencing Capacities at the MPI-MG

3 x Capillary Systems


IT Infrastructure

Long ReadTechnologies

Short ReadTechnologies

TB

GB

25 x 32 (64) Compute Server with 128 (512 GB) RAM4 peta byte Storage Capacity


1. Technologies

Illumina

Roche / 454



Sequence Capture

Amplicon Sequencing

3. Outlook

Technologies


Genome Sequencer FLX HiSeq 2000/SOLiD

• ChipSeq• MeDipSeq• miRNA• RNAseq• Sequencing of target regions• Whole genome resequencing

• de novo Sequencing • Metagenome Analyses• Amplicon Sequencing• Full length Transcriptome Analyses• Sequencing of target regions


Principle Illumina Sequencing

Library Preparation

Attachement of single molecules to surface

Amplification to form clusters

Cluster Generation


5’

G

T

C

A

G

T

C

A

G

T

C

A

GT

3’

5’

C

A

G

TC

A

T

C

A

C

C

TAG

CG

TA

First base incorporated

Cycle 1: Add sequencing reagents

Remove unincorporated bases

Detect signal

Cycle 2-n: Add sequencing reagents and repeat

Sequencing by Synthesis (SBS)


Referenzsequenz ....CGAGCGAATGAAGTCGGGAGTCGTAATGAGCCCGTAATCCCGTTAGTA....

CGAGCGAATGAAGTCGGGAGTCCTAATGAGCCCGTAGAGCGAATGAAGTCGGGAGTCCTAATGAGCCCGTAA

CGAATGAAGTCGGGAGTCCTAATGAGCCCGTAATCCTGAAGTCGGGAGTCCTAATGAGCCCGTAATCCCGTT

TCGGGAGTCCTAATGAGCCCGTAATCCCGTTAGTA

Sequence Reads

Conversion of image data to DNA sequences


Input Material:

~ 1-3 µg DNA shotgun Sequencing~ 10 ng ChipSeq Sequencing

Library Preparation:

~ 1.5 days

Cluster Generation:

~ 1 day

Run Time/

Single read ~ 2 days (36 b)Read Length:

Paired End ~ 10 days (2 x 100 b)

Data Processing:

~ 1 day

Output:

Paired End ~ 500 Gb

Reads:

up to 4800 Mio

Facts Illumina Sequencing (HiSeq 2000)


1. Genome is loaded into a PicoTiter™

plate

3. Load Reagentsin a single rack

4. Sequencing

2. Load PicoTiter plate into instrument

454 Sequencing Instrument


Principle 454 Sequencing

Library Preparation

Emulsion PCR

Depositing DNA Beads into the PicoTiter™Plate

Pyrosequencing

Emulsion Breaking


Input Material:

~ 0.5 µg DNA

Library Preparation :

~ 4 hours

Emulsion PCR:

~ 1 day

Run Time:

20 hours

Data Processing:

~ 10 hours

Output:

Titanium+ 700 -

1000 MB

Reads: Titanium+ 1.000.000 -

1.600.000

Read length:

700 -

800 bases

Facts 454 Sequencing


Sequencing Pipeline

Library Preparation

Library Quantification

Bead EnrichmentSequencing


1. Technologies

Illumina

Roche / 454



Sequence Capture

Amplicon Sequencing

3. Outlook

Projects and Applications


Goals

•

A public database of essentially all SNPs and detectable CNVs with allele frequency >1% in each of multiple human population samples

•

Pioneer and evaluate methods for:• Generating data from next-generation sequencing platforms• Exchanging and combining data and analytical methods• Discovering and genotyping SNPs and CNVs from nextgen data• Imputation with and from next generation sequencing data

•

454, Illumina and AB SOLiD platforms

•

Academic genome centers in US, UK, Germany, China and platform companies

(Nature 2010, Science 2010 and Nature 2011)


OncoTrack, “Methods for systematic

next generation oncology biomarker

development”, is an international consortium of over

60 scientists, that has launched one of

Europe’s largest collaborative academic‐

industry research projects to develop

and assess novel approaches for

identification of new markers for colon

cancer.


DNA RNA

Protein

MethylationMutations

mRNAmiRNACell lines

Tissues

Sequencing

Bioinformatics


RNAseq

expression profiling

total RNA Isolation

quality control

small RNA Depletion

dsDNA generationusing random hexamers

Illumina library preparation

massive parallel sequencing

Mapping


GWAS Candidate Genes

Whole Exome

0.5 –

5 MB 35 MB385 k Array, NimblegenIn-solution Enrichment

2.1 Mio Array, NimblegenIn-solution Enrichment

Sequence Capture


Targeted Resequencing: Project outline

Identification patients Sequence capture

Next-Gen sequencing

“Bioinformatics”Follow-up sequencingFunctional characterization

Work-flow

Sample preparation


Principle of sequence capture

DNA Preparation Enrichment of target regions Sequencing

A1 SP1

A2

genomicDNA

Fragments (200‐500bp)

Ligation of adapters

Hybridization

Selection with

streptavidin beads

Amplification and

Quantification


Cleft lip with or without cleft palate (CL/P)Cooperation with M. Nöthen and E. Mangold

• Prevalence among live births ~ 1 : 1.000

• Risk for siblings 1 : 20 –

1 : 25

• λs

40 -

50

Epidemiology of Epidemiology of nonsyndromic CL/Pnonsyndromic CL/P

Mangold E. et al. (2010), Nature Genetics


•

3 Loci on chr 8 (640Kb), 10 (161Kb) and 17 (340Kb) in 20 affected individuals

•

MID tagging and pooling of 10 samples

•

Enrichment using the 2.1M NimbleGen array

•

Sequencing on a Roche GS FLX system

Cleft lip with or without cleft palate (CL/P)Resequencing as follow up of GWAS


Mapping


•• 6.726 unique variants (>10 x Coverage)

•• 3.783 variants not listed in dbSNP (hg19)

•• 4 coding Variants

•• Detection of structural Variations not yet finished

Cleft lip with or without cleft palate (CL/P)Preliminary Results


Mutation detection pipeline quality

Concordance with Affymetrix Array "genome-wide human SNP array 6.0"


AimDetection and quantification of new and known variants

METHODAmplification and sequencing of target regionsMultiple alignments of sequences against a reference

reference

patient sequences

Amplicon Sequencing


Amplicon Sequencing

B-primer (21 bp)

MID

MID

key

key

A

B

A-primer (21 bp)

Sequence of interest

Locus‐specific PCR

amplification

emPCR Amplification

and sequencing

•

Long reads required to sequence through the locus specific

primer, enable haplotyping over longer distances•

100s to 1000s of amplicon clones sequenced simultaneously


Amplicon Sequencing

IRON StudyInterlaboratory Robustness of NGS


Amplicon Sequencing

IRON StudyHematology Focus Group


Amplicon Sequencing

IRON StudyResults

• per each amplicon, the median coverage eached was 713-fold, ranging from 553-fold to 878-fold

• a total of 92 variants (44 distinct mutations and 10 SNPs) were observed

• in comparison to data available from Sanger sequencing, 454 amplicon deep-sequencing detected all mutations and SNPs that were previously known

• we here confirm in a multicenter analysis that amplicon-

based deep-sequencing is technically feasible, achieves a high concordance across multiple laboratories, and therefore allows a broad and in-depth molecular characterization of hematological malignancies.

Kohlmann et al. (2011), Leukemia


Sensitivity of mutation detection as a function of tumor cell content

Querings et al. (2011), PlosOne


Establishment of small scale NGS systems

Analysis of complete genomes

Personalized medicine

Outlook


Acknowledgments

Hans Lehrach

Bernhard Herrmann

Hilger Ropers

Martin Vingron

Michal SchweigerMartin Kerick

Markus Ralser

Sequencing Facility:Ilona HauenschildSonia PaturejTina MoserIna LehmannNorbert MergesDaniela RothSabrina Rau

Heiner KuhlSven KlagesMartin Werber


Thanksfor your attention!

overview of next generation sequencing platform … 2012/0305 - timmermann.pdf · next generation...

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