VIEWS: 40 PAGES: 34 POSTED ON: 6/26/2011
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
"Ultra-High Throughput DNA Sequencing"