Transcript Genomics Core, Dr. Yuannan Xia

Affymetrix Microarray and
Illumina/ Solexa NextGen Sequencing
Yuannan Xia, Ph.D
Genomics Core Research Facility
10.27.2009
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
Affymetrix Expression Arrays
Array 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
Bridge Amplification of Individual Templates by PCR
Cluster Generation
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
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.
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
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)
Genomic Resequencing
Data of Resequencing of 19 yeast genomes
Nucleus
Chromatin immunoprecipitation sequencing
Crosslink
(ChIP-seq)
IP
Genome-wide analysis
Sequencing
Map binding
sites
• Gene Regulation and Control
• Epigenetic modifications
• DNA-protein interactions
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
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)
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.
ACKNOWLEDGMENTS
Dr. Mike Fromm
Drs. Jean-Jack Riethoven
Ms. Mei Chen