July 7th 2009 - uni-leipzig.de · July 7th 2009 DNA sequencing. Sequencing instruments at MPI EVA 2 x 5 x 454 FLX Titanium Illumina Genome Analyzer ABI SOLiD HeliScope ABI 3730/3730xl

July 7th 2009

DNA sequencing

Sequencing instruments at MPI EVA

5 x2 x

454 FLX Titanium Illumina Genome Analyzer ABI SOLiD HeliScope

ABI 3730/3730xl

pictures by Illumina, Roche, ABI, Helicos

Overview

• Sequencing technologies

• Sequencing strategies

• Sample preparation

Sanger sequencing - principle

= dideoxy method = chain termination method

Template (PCR product, plasmid) dNTP ddNTP

Sanger sequencing - principle

autoradiogram annotated bands

Original method

Sanger sequencing - technology

Improved over time, automated sequencing

- dye-labelled ddNTPs

- capillary electrophoresis +

ABI 3730/3730xl

=

Sanger sequencing – accuracy

Phred-scores = quality scores:

- peak height

- peak shape

- peak density

Sanger sequencing – throughput

200 €

€ run

2 cents

€ base

50-100 kb96500 –1100 b

Sanger

Bases per run

Sequences per run

Read length

Technology

Sanger sequencing – what you need

1) Sample: Clonal copies of your sequencing template

- PCR product

- plasmid

2) Sequencing primer

Sanger sequencing – strategies

A small and simple exon (1 000 bp)

DNA

PCR

PCR product

sequencing2 (4) sequences

A human mitochondrial genome (16 500 bp)

PCRPCR product

sequencing64 sequences


Lysozyme, short exon 1 (500 bp); many paralogues!

DNA

PCRPCR product

subcloning

many sequences

A bush baby mitochondrial genome (16 500 bp); divergent!

LR-PCR LR-PCR product sequencing by

primer walking

64 sequences

sequencing


Genome sequencing

DNA

Chop into pieces

A lot of sequencing,

assembly

Venter style

(WGS) subcloning


Genome sequencing

Consortium style

(hierarchical shotgun)

Lander et al. 2001. Nature 409:860-921

454 sequencing – principle

Pyrosequencing (Nyrén / Ronaghi 1996)

Sequencing by synthesis

- Successive addition of nucleotides (dATPαS,dCTP,dGTP,dTTP)

- Nucleotide incorporation enzymatically translated into light

TACACGACGCTCTTCCGATCT TACACGACGCTCTTCCGATCTAAGTACACGACGCTCTTCCGATCTAATACACGACGCTCTTCCGATCTAAGTTTACACGACGCTCTTCCGATCTAAGTTG

dATPαSdATPαSdATPαSdATPαS

dATPαSdATPαSdCTPdCTPdCTPdCTP

dCTPdCTPdGTPdGTPdGTPdGTP

dGTPdGTPdTTPdTTPdTTPdTTP

dTTPdTTP

GATGTGCTGCGAGAAGGCTAGATTCAACGAGGAGCATTGCACTAGCCTTCTCGAGCATACG


Pyrosequencing massively parallelized by 454 Life Sciences

454 sequencing is not single molecule sequencing

⇒ Parallelization of sample preparation and amplification required

454 Sequenzier-Technologie

I - IIIPreparation of a

sequencing library



Emulsions PCR(emPCR)

IV



V

Bead enrichmentPrimer annealing



Sequenzierung


454 sequencing – accuracy

Phred Q44

Show homopolymer problems

454 sequencing – throughput

2 cents200 €50-100 kb96500 –1100

Sanger

0.001 cents6000 €500 Mb~1 million500454 Titanium

€ run € baseBases per run

Sequences per run

Read length

[bp]

Technology

454 sequencing – applications

Genome sequencing: Sanger

DNA

Chop into pieces

A lot of sequencing,

assembly

Venter style

(shotgun) subcloning

Genome sequencing: 454

DNA

Chop into pieces

less sequencing,

assembly

Venter style

(shotgun) library preparation


Bush baby mitochondrial genome: Sanger

Bush baby mitochondrial genome: 454

LR-PCR LR-PCR product sequencing by

primer walking

LR-PCR LR-PCR product Shotgun sequencing

660 sequences

~ 20x oversampling

64 sequences


PCR product sequencing (Lysozyme): Sanger

PCR product sequencing (Lysozyme): 454

DNA

PCRPCR product

subcloning

many sequences

sequencing

sequencinglibrary preparation

a LOT of sequences

DNA

PCRPCR product

454 sequencing – limitations and solutions

Large amounts of starting material (5 ug)

Meyer et al.; Nucleic Acids Research 2008

quantitative PCRreduces material demands from ~ 5 μg to ~ 20 pg


Sequencing samples in parallel

- Initially limited to 16

GS FLX Titanium platform

- 1/16th lane ~ 25,000 sequences, 500 €

~ 2000 x coverage of a 6 kb plasmid

~ 700 x coverage of a mitochondrial genome

Meyer et al.Nucleic Acids Research 2007Nature Protocols 2008



Using barcoding (e.g. PTS)

- 1/16th lane ~ 25,000 sequences, 500 €

~ 100 plasmids (6 kb) with 20 x coverage

~ 35 mitochondrial genomes with 20 x coverage

~ 1,250 PCR products with 20 x coverage

Limitations in sequencing throughput

=> Limitations in sample preparation

Direct multiplex sequencing

Stiller et al. Genome Research (in press)


Solexa (Illumina) sequencing – principle

Reversible terminator sequencing

Modified polymerase incorporates dye-labeled, terminated nucleotides

1) Incorporation of a single nucleotide

2) Detection of label

3) Removal of terminator/label

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAACACACTCTTTCCCTACACGACGCTCTTCCGATCT

A

C

G

T

AC

G

T

CGTTTTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAAC

ACACTCTTTCCCTACACGACGCTCTTCCGATCT

A

C

G

T

AC

G

T

GTTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAAC

ACACTCTTTCCCTACACGACGCTCTTCCGATCT

A

C

G

T

AC

G

T

T

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAACACACTCTTTCCCTACACGACGCTCTTCCGATCT G

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAACACACTCTTTCCCTACACGACGCTCTTCCGATCT T

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAACACACTCTTTCCCTACACGACGCTCTTCCGATCT CGTT

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAACGTTGCAGGAGCATTGCACTAGCCTTCTCGAGCATACGGCAGAAGACGAACACACTCTTTCCCTACACGACGCTCTTCCGATCT


pictures by Illumina, Inc.

Sodium hydroxide melting

flow cell

pictures by Illumina, Inc.




Solexa (Illumina) sequencing – accuracy

1 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATAAATTA1 AAAAAAAAAAAAACAAAAAACAAAAAAAAAACAAACAAAACAACAAATAA1 AAAAAAAATATTTAATTATTTTTATTTATAATTTTTTTGTTTTTTGTTTT1 AAACAAACCACACAAACAAAAAAACACAACAAAACAACACCACCACCCAA1 ATTCTATTTAATACAAATAAAATATCAATTTAAAACTACACTATACATAA1 CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACA1 CAAATATATTTATATTTATTTTTTTATTTAATTTTTATATTTTTATTTAT1 CATTTATTCTTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTTTTTTT1 CCCCCCCCCCCCCCCCCCCCACCCCCCCCCCACCCACCCCACCCCCCCCC1 CCCCCCCCCCCCCCTTCCCCCCTCTTCTTCTCTCTTTTCTTTTTTTTTTT1 CCCCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT1 CCCGCGCCCCCCCGCCGCCGCGCCCAGCCCAGGCCACCACACACGCACCC1 CCTCCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Artifact sequences

Solexa (Illumina) sequencing – accuracy

1) Map all reads against a reference sequence

2) Eliminate reads with > 2 mismatches in the first 36 bp

3) Check error profiles for the remaining reads

0 10 20 30 40 500 10 20 30 40 50

0.00

0.01

0.02

0.03

Position in read

Mis

mat

ch ra

te

A/CA/GA/TC/AC/GC/TG/AG/CG/TT/AT/CT/GN

Bustard IbisAverage raw error: 2.02% Average raw error: 1.13%

Solexa (Illumina) sequencing – throughput

0.001 cents6,000 €500 Mb~1 million500454 Titanium

0.00004 cents

10,000 €28 Gb~ 140 million

2 x100

Solexa(currently)

2 cents200 €50-100 kb

96500 –1100

Sanger

€ run € baseBases per run

Sequences per run

Read length

[bp]

Technology

Ultra high-throughput sequencing

Solexa (Illumina) sequencing – applications

Genome Re-Sequencing

DNA

Chop into pieces

sequencing

mapping assemblylibrary preparation

8x coverage of human genome

Targeted Sequencing

~ 1 lane of the flowcell ~ 20 million sequences

1 million PCR products

12,500 mitochondrial genomes at 20 x coverage ?

Array capture

Glass slide

Probes

Genome-wide in situ exon capture for selective resequencingHodges et al., Nature Genetics., 2007

• ~5Mb targeted per array•7 arrays, whole exome•~98% of exons retrieved6,000 LR-PCRs

Target enrichment methods

Target enrichment methods

Combine multiplex array capture and sequencing

DNA

shearingpooling

prepare barcoded libraries

DNA

shearing

For each project, array with different targets

Solexa sequencing

How long until we only sequence genomes?

Other sequencing technologies

ABI/SOLiD Polonator Helicos

And dozens under development:

- PacBio

- Oxford Nanopore

- ...

Be warned...

Skills required for DNA sequencing projects

1 % 99 %

Thanks!

For your attention...

MPI EVAN

Martin Kircher

Documents

July 7th 2009 - uni-leipzig.de · July 7th 2009 DNA sequencing. Sequencing instruments at MPI EVA 2 x 5 x 454 FLX Titanium Illumina Genome Analyzer ABI SOLiD HeliScope ABI 3730/3730xl