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Affymetrix Microarray and Illumina/ Solexa NextGen Sequencing Yuannan Xia, Ph.D

Affymetrix Microarray and Illumina/ Solexa NextGen Sequencing Yuannan Xia, Ph.D Genomics Core Research Facility 10.27.2009. Affymetrix GeneChip Microarray System.

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Affymetrix Microarray and Illumina/ Solexa NextGen Sequencing Yuannan Xia, Ph.D

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  1. Affymetrix Microarray and Illumina/ Solexa NextGen Sequencing Yuannan Xia, Ph.D Genomics Core Research Facility 10.27.2009

  2. Affymetrix GeneChip Microarray System 1. High density oligo array: a single array containing 1 – 6 million features generates 1 – 6 million probe hybridization data points for summaring to values of 20K – 50K genes. 2. Hybridization Oven 640: Hybridize samples to array 3. Fluidics Station 450 : washing and staining 4. Scanner 3000 7G: Confocal laser scanning; High pixel resolution at 0.5 micron level 5. Data generating and processing software: GCOS Software pipeline for data mining and annotation: Bioconductor Rosetta, AffyMiner, Ingenuity, Netaffix

  3. Affymetrix Expression Arrays

  4. Illumina/Solexa Genome Analyzer II System Flow cell – A glass slide with 8 channels (lanes) and 16 manifold ports for performing all PCR and sequencing reactions inside each channel. Cluster generation station – Perform PCR bridge amplification to generate clusters inside the channels of flow cell and prepare flow cell ready for sequencing GAII and Paired End Module – Perform sequencing, imaging, cluster modification for paired end read 2 sequencing

  5. Two Key Chemistries used in Solexa Sequencing Technology • PCR bridge amplification of individual templates in a shotgun library to generate clusters (DNA polymerase colony) • High cluster density: 10 – 20 million/Lane • 80 – 150 million/Run • Reversible Terminator Sequencing Chemistry • Allow to incorporate only ONE nucleotide at each cycle • Generate accurate (>99.5%) sequences: • 300 – 800 Megabases/lane • 3 – 6 Gigabases/Run

  6. Bridge Amplification of Individual Templates by PCR

  7. Cluster Generation

  8. Sequencing by Synthesis Using Reversible Terminators >All 4 bases with Reversible Terminators >4 labeling colors >Terminators can be removed >Add all 4 nucleotides in one reaction >No problem with homopolymer repeats >Higher accuracy

  9. Steps of Sequencing by Synthesis A B C D A. Extend first base T, read, and deblock. B & C, Repeat step A to extend strand. D. Generate base calling.

  10. Base Calling From Image Raw Data Read a Cluster-a(xa,ya) Cycles 1 - 9 Cluster-b (xb,yb) Read b The identity of each base of a cluster is read off from sequential images

  11. Solexa Sequencing Applications at UNL • Genomic Resequencing • Yeast genome (V. Gladyshev; AGP Corn Processing) • Fugus genome Aspergillus (S. Harris) • ChIP Sequencing • Arabidopsis CHlP DNA (Fromm; Cerutti) • mRNA Sequencing - Transcritome • Arabidopsis transcriptom (H. Cerutti) • Human KSHV cell transcrptome (C. Wood) • Chlorella/Virus transcriptome (J. Van Etten) • Mole rat transcriptome (V. Gladyshev and D.Fomenko) • Fugus Aspergillus transcriptome (S. Harris) • Paired End Sequencing • Arabidopsis mitochondrial genome (S. Mackenzie) • Small RNA sequencing • Several UNL faculty have expressed strong interest. (Y.Bin, H. Cerutti, J. Mower, J. Alfano)

  12. Genomic Resequencing Data of Resequencing of 19 yeast genomes

  13. Nucleus Crosslink IP Sequencing Map binding sites • Chromatin immunoprecipitation sequencing • (ChIP-seq) • Genome-wide analysis • Gene Regulation and Control • Epigenetic modifications • DNA-protein interactions

  14. Transcriptome Analysis – mRNA-Seq • Relative expression of transcripts • Analysis of splice variants/coding SNPs • Analysis of non-coding RNAs • Transcript discovery

  15. Paired End and Mate Pair Sequencing Provides long range information – Repeat sequences – Characterize copy number variants & rearrangements – De novo assembly Increases output per flow cell

  16. Workflow of the service Genomic DNA, total RNA, CHlP DNA (exp design, QC) DNA shotgun library preparation (SR, PE, cDNA) Cluster generation (35 PCR amplification cycles) Sequencing of clusters on GAII (1 TB machine, sequencing, imaging, image processing, base calling) Data analysis on remote server at Bioinfornatics Core Facility (8 TB machine, base calling , read alignment using Illumina pipeline software)

  17. Cost of Gene Expression Profiling • Microarray • $500 - $650/array/sample • $3000 - $4000 for 2 treatments-3replicats 6 samples experiment • (6 arrays) • Illumina • $1300/lane/sample (400Mb sequence), $2100/2 lanes/sample (800Mb) • $ 4200 for 2 treatment 2 samples 4 FC lanes experiment • (without replicates)

  18. Challenges More than 40 billion nucleotide sequences have been generated, will be double soon. Need solutions to - Sample preparation (e.g small RNA libraries, CHlP pulldown) - Further extracting sequencing data - Biological annotations - Data storage and management - Drafting publications Budget: - Maintaining both Affymetrix System and Illumina Solexa System is expensive. - Cost for upgrading the system.

  19. ACKNOWLEDGMENTS Dr. Mike Fromm Drs. Jean-Jack Riethoven Ms. Mei Chen

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