Documents
Resources
Learning Center
Upload
Plans & pricing Sign in
Sign Out

Ultra-High Throughput DNA Sequencing

VIEWS: 40 PAGES: 34

									 Ultra-High Throughput DNA
Sequencing on the 454/Roche
           GS-FLX
  Methods, Automation, Applications

           Graham Wiley
             Roe Lab
                                      1
                                                 100,000,000
                            454/Roche GS-FLX

A Brief History of Automated
DNA Sequencing Instruments
                                     454-GS20   64,000,000




                          ABI 3730

     ABI       ABI 3700
     370/377

                                                2007
                                                       2
     454 GSFLX Sequencer
• Pico-scale
  sequencing
  reactions
• 2 Core
  Techniques:
  – Emulsion PCR
  – Pyrosequencing



                           3
              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.




                                                         4
Emulsion PCR




               5
         Load Beads into 454 Plate
                      Load Enzyme
Load beads into          Beads
PicoTiterPlate


                      Centrifugation




          44 μm




                                       6
                              Pyrosequencing
                                               dTTP
                              •Polymerase adds (1)                                           Polymerase
                              nucleotide (dNTP)
DNA                   A   A   T   C   G   G   C   A   T   G   C    T   A   A    A   A    G   T   C   A


Bead                                                      APS                  PPi
                                                                                                     T


                                                                                                         Annealed Primer


                                                                               (2) •Pyrophosphate
                                                                                        is released (PPi)
               Sulfurylase                        ATP (3)
 Enzyme Bead                                                      •Sulfurylase creates ATP
               Luciferase                                          from PPi and APS


  (5) CCD camera                                          luciferin (4)
     detects bursts                                                            • Luciferase hydrolyses ATP
     of light
                                                                                to oxidize luciferin and
                          Light + oxy luciferin                                 produce light

                                                                                                            7
Pyrosequencing Output




                        8
Base Calling via Flowgram



                      TTCTGCGAA




                             9
         Types of Libraries
• 454/Roche
  – Shotgun
    • Random 250+bp reads
  – Paired-End
    • 25-50bp ends of a circularized DNA molecule
  – Amplicon
    • PCR product for SNP discovery
• Roe Lab
  – Paired-End/Shotgun
    • Best of both worlds
                                                    10
454 Shotgun Library Preparation
      Protocol Overview
      Nebulization



                                  5’                3’
     DNA End Repair
                                   3’               5’



                                 5’            3’
  Adaptor Ligation (A&B)
                                      3’                 5’
                                      A             B

                           5’                         3’
     DNA End Repair                   A    B
                            3’                       5’




  Library Quantification
        on Caliper
                                                              11
454 Paired End/Shotgun DNA
Preparation Protocol Overview
      Shear to 2-4 Kbp fragments
          on the Hydroshear

    Quantitate on Caliper AMS-90


  DNA End Repair & Linker Ligation

Cleave the Terminal Linkers with EcoR1


    Ligate to Circularized the DNA




     Shear to ~500 bp fragments
           in the Nebulizer              12
   454 Paired End/Shotgun DNA
Preparation Protocol Overview (cont)
     Quantitate on Caliper AMS-90



   DNA End Repair, Adaptor Ligation,
         Adapter End Repair



        Amplification (emPCR)



          Pyrosequencing on
          454/Roche GS-FLX             13
 454 Paired-End/Shotgun Assembly
             Process
• 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
  – ~3-5% of the total reads contain the middle linker
    sequence
• Assembly of the reads by Newbler
• Convert paired ends for Exgap ordering and
  orienting
  – *.454f and *.454r
                                                     14
  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
                                                             15
      SPRI Bead Technology




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


• 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                     16
DNA Purification through the Qiagen Minelute
Columns vs Agencourt SPRI Magnetic Beads

  Qiagen Minelute centrifuge column                    Agencourt SPRI magnetic beads




Both procedures give an almost similar yield but the yield is slightly better with the SPRI
beads and the automation of the SPRI bead prep is somewhat easier to achieve
                                                                                        17
  96 well Magnetic Plate for
Purification of the SPRI Beads




                            18
Enzyme Chilling Station




                          19
Zymark SciClone Deck Arrangement



Waste     EtOH      Magnet




Enzyme
Mixes     Shaker             Shaker
           Sample




Buffers                       SPRI
          Shaker
                              Beads

                                      20
Adding SPRI Beads on the
        SciClone




                           21
Magnetically Separating SPRI
  Beads on the SciClone




                           22
Washing SPRI Beads
 on the SciClone




                     23
              Applications
• Whole Genome Sequencing
• Sample Pools
  – BACs
  – Viruses
• EST Libraries
• Bacterial Communities


                             24
    Plant Viruses of the Tallgrass
               Prairie
•   Single or double stranded RNA
•   Typically <10,000bp, ~12,000bp max.
•   4-12 encoded genes
•   Inherent instability of RNA leads to large
    amount of mutations, hence, large
    species variation



                                            25
     cDNA pooling strategy
• Tags on PCR primers allow for
  deconvolution of viral sequences post
  sequencing
• cDNA samples are pooled in sets of 24-
  96 at the Noble Foundation and sent to
  OU for sequencing


                                       26
     Strategy for preparing cDNA ready
      for 454 sequencing from dsRNA
                 5’                                                           3’
                 3’                                                           5’
                                                   Anneal with Random Hexamer Primers followed by
                                                   Reverse Transcriptase PCR Reaction
       5’        5’                                                    NNNNNN 3’



                      NNNNNN
                                               +
                 3’                                                        5’             5’
       5’                                        Additional Rounds of RT PCR
                                                 with Random Hexamer Primers
                      NNNNNN                                                                   3’
                                                                       NNNNNN
                                                                                               5’
      5’              NNNNNN
                                               +
                                                                      NNNNNN
      3’
        RNAse Treatment to Remove any Excess Random Hexamer Primers
        followed by a Taq Polymerase PCR with one of the 20 Tagged Primers
      5’              NNNNNN                                                   GGAAGCCTAGGAGG   3’
      3’                                                                                        5’
                                                                               CCTCCTAGGCTTCCGAGA

3’     GGAAGCCTAGGAGG
                                               +                      NNNNNN                5’
5’ AGAGCCTTCGGATCCTCC                                                                       3’
                                                 Amplified Product Ready for               5’
                                                 Ligating 454 A and B Primers
    AGAGCCTTCGGATCCTCC                                                                          27
A                                                                          CCTCCTAGGCTTCCGAGA       B
   Uniquely Tagged cDNA Sample from the TGP on the 454




                                RT-PCR Sequence




                                      TGP common primer
454 tag
                     TGP Unique tag
(TCAG)                                (CCTTCGGATCCTCC)
                     (GACA)
                                                          28
                      Putative New Allexivirus
                                                 Membrane
                                                 Protein Hypothetical Coat Nucleic Acid
(+)ssRNA        Replicase                  Helicase      Protein      Protein Binding Protein

                                                                                    ~8.5KB
           13               76                      65       74           69
                105              75
                      81              95      83

  05TGP00120




   • BlastX shows a large number of contigs have homology to
     viruses of the genus Allexiviridae
   • Contig sequence lengths cover ~66% of a typical Allexivirus
     genome of ~8.5KB
   • 5 of the 6 genes encoded by Allexiviridae species are
                                                                                       29
     represented in the sequenced contigs
        Current BAC Pooling Strategy
• 10x10 Grid of 100 BAC clones                     Pool A

• 1-fold coverage of each pool of 10
  150Kb BACs is 1.5 Mb
                                          Pool B    X
• 1 quarter 454/Roche GS-FLX
  picotiter plate give ~13Mb or 10-fold
  cov.
• 5 full picotiter plate runs are required
  for 20-fold coverage of each
  individual BAC at the
  horizontal/vertical intersect.
• $12k/run = ~$600/BAC
• Additional ABI 3730 runs are needed
  for each pool to aid in deconvolution
  at ~ $1000 for each of the 20 pools
  and an additional ~$800/BAC or
  $1400 total cost per BAC

                                                            30
Future Tagged BAC Pooling
         Strategy
• 24 uniquely tagged individual shotgun libraries
  would be pooled and sequenced on one full
  454/Roche GS-FLX picotiter plate
• 24 150 Kb BACs would require 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
• At $12k/run = ~$500 per BAC for >20-fold shotgun
  coverage and no ABI 3730 runs are needed to
  deconvolute the individual BACs as each BAC is
  individually tagged                                31
                Conclusions
• It is possible to incorporate both shotgun and
  paired end reads in the same library
• Qiagen Minelute centrifuge columns may be
  replaced by Agencourt SPRI beads after
  enzymatic steps in 454 library preparation.
• The replacement of centrifuge columns with
  magnetic beads as well as the manufacture
  of an enzyme chilling station allows for the
  automation of the library making process
• Through the use of tagged RT-PCR samples
  it is possible to sequence putatively novel
  plant viruses
                                                   32
        Acknowledgments
• Dr. Roe
• Loaders & Data Analyzers: Simone Macmil,
  Doug White, Steve Kenton
• Makers & Breakers: Chunmei Qu, Ping
  Wang, Yanbo Xing, Baifeng Qin, Keqin Wang
• All other members of the Roe lab
• Collaborators
  – OSU: Ulrich Melcher, Vijay Muthamukar
  – Noble Foundation: Marilyn Roossinck, Guoan
    Shen, Byoung Min, Rick Nelson, Tracy Feldman
                                                   33
34

								
To top