Post on 20-Dec-2015
transcript
Affymetrix Microarray and Illumina/ Solexa NextGen Sequencing
Yuannan Xia, Ph.D
Genomics Core Research Facility10.27.2009
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
Affymetrix GeneChip Microarray System
Affymetrix Expression ArraysArray type Number of transcripts Number of genes
Human U133 plus 2.0
>47,000 38,500
Mouse 430 2.0 >39,000 >34,000
Rat 230 2.0 >31,000 >28,000
Arabidopsis ATH1 22,500 >24,000
Drosophila 18,500 18,000
Wheat 61,127 55,052
Rice 51,279
Soybean 58,000 58,000
Barley 25,500 22,000
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
>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
A B C D
Base Calling From Image Raw Data
The identity of each base of a cluster is read off from sequential images
Cluster-a (xa,ya)
Cluster-b (xb,yb)
Read a
Read b
Cycles 1 - 9
Genomic ResequencingYeast genome (V. Gladyshev; AGP Corn
Processing)Fugus genome Aspergillus (S. Harris)
ChIP SequencingArabidopsis CHlP DNA (Fromm; Cerutti)
mRNA Sequencing - TranscritomeArabidopsis 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 SequencingArabidopsis mitochondrial genome (S. Mackenzie)
Small RNA sequencingSeveral UNL faculty have expressed strong interest. (Y.Bin, H. Cerutti, J. Mower, J. Alfano)
Solexa Sequencing Applications at UNL
Chromatin immunoprecipitation sequencing
(ChIP-seq)
Genome-wide analysis• Gene Regulation and Control• Epigenetic modifications• DNA-protein interactions
Nucleus
Crosslink
IP
Sequencing
Map bindingsites
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 assemblyIncreases 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
• $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)
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.