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Massively Parallel High Throughput DNA Sequencing: Automation for Microbial Community, Gene Expression and de novo Deciphering of New Genomes. Bruce A. Roe, Ph.D.,

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Bruce a roe ph d

Massively Parallel High Throughput DNA Sequencing: Automation for Microbial Community, Gene Expression and de novo Deciphering of New Genomes

Bruce A. Roe, Ph.D.,

George Lynn Cross Research Professor of Chemistry and Biochemistry, Advanced Center for Genome Technology, Stephenson Research
and Technology Center, University of Oklahoma


A brief history of long re ad automated dna sequencing instruments abi and 454 roche

454/Roche GS-FLX-XLR

1Gb/run

100

454/Roche GS-FLX

100Mb/run

454-GS20

30Mb/run

75

50

~

~

40

30

20

# Million Bases/Run

10

~

~

2.0

ABI 3730 1 Mb/run

1.5

1.0

ABI 3700 200Kb/run

ABI 370/377 40Kb/run

0.5

0.04

2008

2007

1994

1996

1998

2000

2002

2004

2006

Date of Introduction

A Brief History of Long Read Automated DNA Sequencing Instruments: ABI and 454/Roche


454 gs flx sequencer

454 GS-FLX Sequencer

  • Pico-scale sequencing reactions

  • 2 Core Techniques:

    • Emulsion PCR

    • Pyrosequencing


Emulsion pcr

Emulsion PCR

  • Micro-reactors

    • Water-in-oil emulsion generates millions of micelles.

    • Each micelle contains all reagents/templates for a PCR reaction.

    • ~10 Million individual PCR reactions in a single tube.


Emulsion pcr1

Emulsion PCR


Load beads into 454 picotiter plate

44 μm

Load Beads into 454 Picotiter Plate

Load Enzyme Beads

Load beads into PicoTiter Plate

Centrifugation


Pyrosequencing

DNA

Bead

dTTP

  • DNA Polymerase adds

  • nucleotide (dNTP)

DNA Polymerase

(1)

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

T

PP

APS

Annealed Primer

i

(2)

  • Pyrophosphate

  • is released (PPi)

Sulfurylase

Luciferase

ATP

(3)

  • Sulfurylase creates ATP

  • from PPi and APS

Enzyme Bead

(5)

luciferin

(4)

CCD camera detects bursts of light

  • Luciferase hydrolyses ATP

  • to oxidize luciferin and

  • produce light

Light + oxy luciferin

Pyrosequencing


Pyrosequencing output

Pyrosequencing Output


Base calling via flowgram

Base Calling via Flowgram

TTCTGCGAA


Types of libraries

Types of Libraries

  • 454/Roche

    • Shotgun

      • Random 250+bp reads

    • Paired-End

      • 25-250bp ends of a circularized DNA molecule

    • Amplicon

      • PCR product for SNP discovery

  • Roe Lab

    • Combined Paired-End and Shotgun approach

      • Best of both worlds


Our combined paired end shotgun dna preparation protocol overview

Shear to 2-4 Kbp fragments on the Hydroshear

Quantitate on Caliper AMS-90 or by RealTime PCR

DNA End Repair & Linker Ligation as in paired-end protocol

Cleave the Terminal Linkers with EcoR1

Ligate to Circularized the DNA

Shear to ~500 bp fragments in the Nebulizer but eliminate the enrichment step for fragments containing linker

Our Combined Paired End & Shotgun DNA Preparation Protocol Overview


Our combined paired end shotgun dna preparation protocol overview cont

Quantitate on Caliper AMS-90 or by RealTime PCR

DNA End Repair, Adaptor Ligation,

Adapter End Repair

Amplification (emPCR)

Pyrosequencing of the combined linker-containing (paired end) and shotgun fragments on 454/Roche GS-FLX

Our Combined Paired End/Shotgun DNA Preparation Protocol Overview (cont)


Assembly of sequence reads from our combined paired end shotgun protocol

Assembly of Sequence Reads from Our Combined Paired-End/Shotgun Protocol

  • Separate based on inclusion or exclusion of middle linker

    • Those sequences containing a middle linker are further separated based on the length of the read to either end of the linker sequence

    • ~15% of the total reads contain the middle linker sequence

  • Assembly of the reads by Newbler

  • Convert paired ends for ordering and orienting

    • *.454f and *.454r


Automation of the shotgun library preparation steps

Automation of the Shotgun Library Preparation Steps

  • Why automate?

    • Time

    • Reproducibility

  • What are the obstacles?

    • Reaction Cleanup

      • Qiagen Minelute centrifuge columns are difficult to automate, so replace those steps with

      • Agencourt SPRI magnetic beads and add a magnetic station to the Zymark SciClone bed

    • Enzyme Stability and Storage

      • Build an enzyme cooling station on the Zymark SciClone bed


Spri bead technology

SPRI Bead Technology

  • Solid Phase Reversible Immobilization

  • Carboxyl coated magnetic particles suspended in a solution of 10% PEG and 1.25M NaCl

  • Reversibly binds DNA

    • Hawkins, et al. (1994) DNA purification and isolation using a solid-phase. Nucleic Acids Research, 22(21):4543-4544

http://www.agencourt.com/products/spri_reagents/ampure/


Dna purification through the qiagen minelute columns vs agencourt spri magnetic beads

DNA Purification through the Qiagen Minelute Columns vs... Agencourt SPRI Magnetic Beads

Agencourt SPRI magnetic beads

Qiagen Minelute centrifuge column

At least a 30% increase in yield with the SPRI beads and it is easier to automate when using the SPRI beads


Homemade 96 well magnetic plate for purification of the spri beads

Homemade 96 well Magnetic Plate for Purification of the SPRI Beads

Inverted 96 well DNA sequencing plate with cylindrical magnets


Enzyme chilling station

Enzyme Chilling Station

Plastic rack fitted with Swagelock fittings and tubing for cooling.


Zymark sciclone deck arrangement

Enzyme Mixes

Waste

EtOH

Buffers

Magnet

Zymark SciClone Deck Arrangement

Shaker

Shaker

Sample

SPRI Beads

Shaker


Automated library making on the caliper zymark sciclone

Automated Library Making on the Caliper-Zymark SciClone

To view this automation, get our quicktime movie 454ZymarkPrep.mov


Bruce a roe ph d

We also have increased the average read lengths from 250 to > 315 bases by increasing the number of flows and amounts of reagents

  • Slightly dilute the Substrate, Inhibitor and Apyrase by transferring 2.5mL from one of the Buffer CB bottles to each respective tube in the reagent tube-tray

  • Add 174ul (as opposed to 164ul) from the tube of apyrase to the apyrase buffer tube in the reagent tube-tray.

  • Transfer 150ml Buffer CB from bottle 3 (at the back of the cassette) to bottle 0 (at the front of the cassette).

  • Modify the run script to allow for 130 flow cycles


Bruce a roe ph d

Reuse the Pico Titer plate after cleaning by sonication


Summary methods

Summary - Methods

  • For library preparation, It is possible to:

    • incorporate both shotgun and paired end reads in the same library

    • replace the Qiagen Minelute centrifuge columns with Agencourt SPRI beads in the library preparation and build (or buy) an enzyme chilling station to facilitate automating the library making process

    • eliminate the steps involved in single stranded DNA preparation steps

  • It also is possible to:

    • break the emulsion after emPCR using centrifugation rather than using a Swinlock filter containing a sieving fabric.

    • Increase the volumes of the FLX reagents and increase the number of cycles results in a significantly increased read length.

    • reuse the PicoTiter plate after cleaning by sonication

  • All our protocols are available on our lab protocol web site at url: http://www.genome.ou.edu/proto.html


Applications

Applications

  • Whole Genome Sequencing

  • Pooled samples

    • Plant viruses

    • Plant fungi

    • BAC-based genomic sequencing

  • EST Libraries

  • Bacterial Communities


Novel cdna pooling strategy

Novel cDNA pooling strategy

  • Add tags to the PCR primer sequences to allow for deconvolution of viral sequences post sequencing

  • cDNA samples are pooled in sets with 24 unique individual tags after a two step PCR


Strategy for preparing cdna ready for 454 sequencing from dsrna

5’ 3’

3’ 5’

NNNNNN

CCTTCGGATCCTCC

CCTCCTAGGCTTCC

NNNNNN

CCTTCGGATCCTCC

NNNNNN

CCTCCTAGGCTTCC

NNNNNN

NNNNNN

CCTCCTAGGCTTCC

Strategy for preparing cDNA ready for 454 sequencing from dsRNA

Anneal with Random Hexamer Primers followed by Reverse Transcriptase PCR Reaction

5’

5’

3’

+

5’

5’

3’

5’

Additional Rounds of RT PCR with Random Hexamer Primers

3’ 5’

+

5’ 3’

CCTTCGGATCCTCC

NNNNNN

RNAse Treatment to Remove any Excess Random Hexamer Primers followed by a second Taq Polymerase PCR with one of the 24 four base Tagged Primers

3’ 5’

5’ 3’

CCTTCGGATCCTCC

GGAAGCCTAGGAGG

NNNNNN

CCTCCTAGGCTTCCGAGA

+

3’ 5’

5’ 3’

CCTCCTAGGCTTCC

NNNNNN

GGAAGCCTAGGAGG

AGAGCCTTCGGATCCTCC

Amplified Product Ready for Ligating 454 A and B Primers

5’

A

AGAGCCTTCGGATCCTCC

B

CCTCCTAGGCTTCCGAGA


Uniquely tagged cdna sample on the 454

RT-PCR Sequence

TGP common primer

(CCTTCGGATCCTCC)

454 tag (TCAG)

TGP Unique tag (GACA)

Uniquely Tagged cDNA Sample on the 454


Bruce a roe ph d

10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome Isolation

S.pombe

Po OkAlf-8 in all 4 lanes

  • Excise individual chromosomal bands, freeze at -200C and then melt by heating to 65 0C.

  • Mix 500 ul aliquots of TE saturated phenol and melted gel and re-freeze at -200C

  • Centrifuge at 2500 RPM in a table top centrifuge at -200C

  • Remove aqueous layer and extract any residual phenol twice with water-saturated ether

  • Ppt with 2.5 vol of 95% ethanol/acetate, wash 70% ethanol and dry the DNA

  • Dissolve the DNA in 10 ul of 10:0.1 TE

Chr. #

1

2

3

5.7 Mb

4

4.6 Mb

5

6

7

3.5 Mb


10 day contour clamped homogenous electrophoretic field chef gels for chromosome isolation

Eluted & amplified chromosomes on a 1% agarose gel

BAC Hind3 1 2 3 4 5 6 7 Hind3

  • Qiagen REPLI-g Mini kit was used to amplify the chromosomes

  • 2.5 ul of the purified chromosomal DNA was mixed with 2.5 ul of Qiagen denaturation buffer for 3 minutes at 250C followed by mixing with 5ul of Qiagen neutralization buffer.

  • A master mix containing 10 ul nuclease-free water, 29 ul reaction buffer (containing dNTPs and exonuclease-resistant primers) and 1 ul of the Qiagen’s DNA polymerase was added to the treated chromosomal DNA and incubated at 300C overnight.

  • The amplified chromosomal DNA product then was verified on a 1% agarose gel by electrophoresis and subjected to the mixed shotgun paired-end sequencing where over 90% of the sequences matched in our CRR database

10 Day Contour Clamped Homogenous Electrophoretic Field (CHEF) Gels for Chromosome Isolation


Bruce a roe ph d

Summary of our use of CHEF gels for chromosome isolation and subsequent amplification for sequencing

  • Using our long established freeze/thaw phenol extraction protocol, individual chromosomes can be purified from chromosome grade agarose CHEF gels and then

  • Amplified using the Qiagen REPLI-g Mini kit

  • Sequence data can obtained after library making, emPCR and massively parallel pyrosequencing on the 454/Roche GS-FLX with over 90% of the sequences matching our target genome/fungal database


Strategy of adding the 454 roche mid based tags prior to bac pooling

BAC growth in 96 deep well microtiter plates

Robotic BAC isolation via the cleared lysate protocol using the Hydra robot.

Sheer each BAC individually and create the paired end libraries on the Zymark SciClone robot.

Individually tagged A linkers are added with B linkers prior to pooling 12 tagged libraries, followed by

emPCR, and half-plate sequencing of each pool.

Strategy of adding the 454/Roche MID-based Tags prior to BAC Pooling


Strategy of adding the 454 roche mid based tags prior to bac pooling1

Strategy of adding the 454/Roche MID-based Tags prior to BAC Pooling

12 uniquely tagged individual shotgun libraries would be pooled and sequenced on each half- 454/Roche GS-FLX picotiter plate, 24 tagged libraries/full plate

24 150 Kb BACs requires 3.6 Mb for 1 x sequence coverage

With >75 Mb of DNA sequence obtained per full plate, >20x coverage is obtained for each of the 24 pooled BACs

96 BACs would therefore require 4 full plate runs on the 454/Roche GS-FLX and no ABI 3730 runs are needed to deconvolute the individual BACs as each BAC is individually tagged

The BACs then are easily closed and finished using PCR-based methods.


Analysis of ordered and oriented combined shotgun paired end results

Analysis of ordered and oriented combined shotgun/paired-end results

vector

repeat sequences missing in the 454 data but present in the 3730 and/or obtained by PCR-based closure

454/Roche GS-FLX only assembled sequences

Phrap-assembled ABI-3730 and 454/Roche GS-FLX sequences

Un-joined 454 data often with no missing base but joined by 454 paired-ends and spanned by 3730 or PCR-based sequences

Our present strategy is to use the combined shotgun/paired-end pyrosequencing approach on the 454/Roche GS-FLX followed by PCR-based closure methods.


Acknowledgments

Acknowledgments

  • Collaborators

    • Plant Virus studies

      • Oklahoma State University: Ulrich Melcher, Vijay Muthamukar

      • Noble Foundation: Marilyn Roossinck, Guoan Shen, Byoung Min, Rick Nelson, Tracy Feldman

    • Phymatotrichopsis omnivora aka Cotton Root Rot Fungi

      • Oklahoma State University: Steve Marek

      • Noble Foundation: Carolyn Young

    • Medicago truncatula

      • University of Minnesota: Nevin Young, Roxanne Denny, Steven Cannon (now at Iowa State), Arvind Bhari, Shelly Wang

      • The JCV Institute: Chris Town, Foo Cheung

      • The John Innes Institute, UK: Giles Oldroyd & Sanger Institute: Jane Rogers

      • Toulouse/INRA & Genoscope, France: Fredric Debelle, Francis Quetier

      • Munich Bioinformatics Center IMGAG Consortium: Claus Mayer

  • Funding from the NSF Plant Genome, Microbial and EPSCoR Programs and the USDA


Bruce a roe ph d

OU Genome Center Personnel

Automation

Graham

Fares

Doug

Simone

Nature gives up her secrets to the prepared mind, driving innovation

www.genome.ou.edu/proto/htmll


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