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

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 G: 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 Affymetrix GeneChip Microarray System

Affymetrix Expression Arrays Array typeNumber of transcriptsNumber of genes Human U133 plus 2.0 >47,00038,500 Mouse >39,000>34,000 Rat >31,000>28,000 Arabidopsis ATH122,500>24,000 Drosophila18,50018,000 Wheat61,12755,052 Rice51,279 Soybean58,000 Barley25,50022,000

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

Two Key Chemistries used in Solexa Sequencing Technology 1.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 2.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

Bridge Amplification of Individual Templates by PCR

Cluster Generation

>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 Sequencing by Synthesis Using Reversible Terminators

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

Base Calling From Image Raw Data The identity of each base of a cluster is read off from sequential images Cluster-a (x a,y a ) Cluster-b (x b,y b ) Read a Read b Cycles 1 - 9

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) Solexa Sequencing Applications at UNL

Genomic Resequencing Data of Resequencing of 19 yeast genomes

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

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

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

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) Workflow of the service

Cost of Gene Expression Profiling Microarray $500 - $650/array/sample $ $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)

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.

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