microarrays and other high-throughput methods bmi/cs 576 colin dewey fall 2010
DESCRIPTION
More High-Throughput Methods 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 specTRANSCRIPT
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Microarrays and Other High-Throughput Methods
BMI/CS 576www.biostat.wisc.edu/bmi576/
Colin [email protected]
Fall 2010
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One High-Throughput Method: Microarrays• one common HT study type: measuring RNA abundances
• 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
genes
mRNAs
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More High-Throughput Methodsvaried: individuals, strains, cell types, environmental conditions, disease states, etc.measured: RNA quantitiestechnology: microarrays, RNA-seqvaried: same as abovemeasured: protein quantitiestechnology: 2D gel electrophoresis + mass spec
varied: same as abovemeasured: small molecule quantitiestechnology: 2D gel electrophoresis + mass spec
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More High-Throughput Methods
varied: single (or pairs) genes knocked outmeasured: some “reporter” quantity of interesttechnology: deletion libraries
varied: individualsmeasured: variation at specific genome locationstechnology: SNP chips, next-generation sequencing
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High-Throughput Methods for Detecting Interactions
varied: cell types, environmental conditions etc.measured: protein-DNA interactionstechnology: chip-ChIP, ChIP-Seq
varied:measured: protein-protein interactionstechnology: two-hybrid systems, mass spec
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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.
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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
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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
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Complementary Hybridization
AGCGGTTCGAATACC
TCGCGAAGCTAGACA
CCGAAATAGCCAGTA
UCGCCAAGCUUAUGG
• due to Watson-Crick base pairing, complementary single-stranded DNA/RNA molecules hybridize (bond to each other)
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Complementary Hybridization
AGCGGTTCGAATACC
• 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
UCGCCAAGCUUAUGG
TCGCCAAGCTTATGG actual gene
cDNA
mRNA
reverse transcriptase
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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
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Spotted Arrays• two samples (reference and test) of mRNA are reverse
transcribed to cDNA, labeled with fluorescent dyes and allowed to hybridize to array
test
reference
mRNA cDNA
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Spotted Arrays• lasers applied to the arrays yield an emission for each
fluorescent dye
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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
i
iiG
greenredlog=
where red is the test expression level, and green is the reference level for gene G in the i th experiment
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Oligonucleotide Arrays• most common are Affymetrix’s GeneChips™
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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
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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
gene
25-mers
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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
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RNA-Seq
• Sequencing technology is rapidly advancing• Idea: sequencing is so cheap we can directly
sequence mRNAs• “digital gene expression”
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RNA-Seq
Mortazavi et al., Nature Methods, 2008
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RNA-Seq vs. Microarrays
Mortazavi et al., Nature Methods, 2008
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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 …