Microarrays and Other High-Throughput Methods

1. Microarrays and Other High-Throughput Methods BMI/CS 576 www.biostat.wisc.edu/bmi576/ Colin Dewey cdewey@biostat.wisc.edu Fall 2010

2. One High-Throughput Method: Microarrays • one common HT study type: measuring RNA abundances mRNAs genes • what is varied: individuals, strains, cell types, environmental conditions, disease states, etc. • what is measured: RNA quantities for thousands of genes, exons or other transcribed sequences

3. varied: individuals, strains, cell types, environmental conditions, disease states, etc. measured: RNA quantities technology: microarrays, RNA-seq varied: same as above measured: protein quantities technology: 2D gel electrophoresis + mass spec varied: same as above measured: small molecule quantities technology: 2D gel electrophoresis + mass spec More High-Throughput Methods

4. varied: single (or pairs) genes knocked out measured: some “reporter” quantity of interest technology: deletion libraries    varied: individuals measured: variation at specific genome locations technology: SNP chips, next-generation sequencing More High-Throughput Methods

5. varied: cell types, environmental conditions etc. measured: protein-DNA interactions technology: chip-ChIP, ChIP-Seq varied: measured: protein-protein interactions technology: two-hybrid systems, mass spec High-Throughput Methods for Detecting Interactions

6. HT Output • for most of these methods, we can think of the output as a 2D matrix in which • rows are gene/protein/small-molecule measurements • columns are individuals/strains/cell-types/treatments etc.

7. Microarrays • microarrays provide a tool for answering a wide range of questions about the dynamics of cells • how active are various genes in different cell/tissue types? • how does the activity level of various genes change under different conditions? • stages of a cell cycle • environmental conditions • disease states • knockout experiments • what genes seem to be regulated together? • can also be used to answer questions about static properties (e.g. genotyping), but we’ll focus on the former class of questions

8. Microarrays • a.k.a. DNA chips, gene chips, DNA arrays etc. • two general types that are popular • spotted arrays (pioneered by Pat Brown @ Stanford) • oligonucleotide arrays (pioneered by Affymetrix Inc.) • both based on the same basic principles • anchoring pieces of DNA to glass/nylon/silicon slides • complementary hybridization

9. AGCGGTTCGAATACC TCGCGAAGCTAGACA CCGAAATAGCCAGTA Complementary Hybridization • due to Watson-Crick base pairing, complementary single-stranded DNA/RNA molecules hybridize (bond to each other) UCGCCAAGCUUAUGG

10. TCGCCAAGCTTATGG Complementary Hybridization • one way to do it in practice • put (a large part of ) the actual gene sequence on array • convert mRNA to cDNA using reverse transcriptase actual gene AGCGGTTCGAATACC cDNA reverse transcriptase UCGCCAAGCUUAUGG mRNA

11. Spotted Arrays • robot puts little spots of DNA on glass slides • each spot is DNA analog of one of the mRNAs we want to measure

12. Spotted Arrays • two samples (reference and test) of mRNA are reverse transcribed to cDNA, labeled with fluorescent dyes and allowed to hybridize to array reference test mRNA cDNA

13. Spotted Arrays • lasers applied to the arrays yield an emission for each fluorescent dye

15. Spotted Arrays • we can’t detect the absolute amount of mRNA present for a given gene, but we can measure amount relative to a reference sample • typically we have a set of measurements • where red is the test expression level, and green is the reference level for gene G in the i th experiment

16. Oligonucleotide Arrays • most common are Affymetrix’s GeneChips™

17. Oligonucleotide Arrays • instead of putting entire genes on an array, put sets of DNA oligonucleotides (fixed length sequences, typically 25-60 nucleotides in length) • oligos are synthesized on the chip • Affymetrix uses a photolithography process similar to that used to make semiconductor chips • there are other processes; Nimblegen (in Madison) uses an array of 786,000 tiny mirrors + photo deposition chemistry • mRNA samples are processed separately on individual arrays, instead of in pairs

18. gene 25-mers Oligonucleotide Arrays • given a gene to be measured, select different n-mers for the gene • can also select n-mers for noncoding regions of the genome • selection criteria • specificity • hybridization properties • ease of manufacturing

19. Oligonucleotide Arrays • put each of these n-mers on the chip • Affymetrix also puts a slight variant (that differs only at the middle base) of each next to it • this supposedly helps factor out false hybridizations • the measurements for a gene are derived from these separate n-mer measurements • present/absent calls • numerical quantity proportional to amount of mRNA present

20. RNA-Seq • Sequencing technology is rapidly advancing • Idea: sequencing is so cheap we can directly sequence mRNAs • “digital gene expression”

21. RNA-Seq Mortazavi et al., Nature Methods, 2008

22. RNA-Seq vs. Microarrays Mortazavi et al., Nature Methods, 2008

23. Several Computational Tasks • identifying differential expression: which genes have different expression levels across two groups • clustering genes: which genes seem to be regulated together • clustering samples: which treatments/individuals have similar profiles • classifying genes: to which functional class does a given gene belong • classifying samples: to which class does a given sample belong • e.g., does this patient have ALL or AML • e.g., does this chemical act like an AHR agonist, or a PCB or …