1. The virochip (UCSF) is a spotted microarray. Hybridization of a clinical RNA (cDNA) sample can identify specific viral expression

2. Figure 2 Supervised classification on prognosis signatures. a, Use of prognostic reporter genes to identify optimally two types of disease outcome from 78 sporadic breast tumours into a poor prognosis and good prognosis group (for patient data see Supplementary Information Table S1). b, Expression data matrix of 70 prognostic marker genes from tumours of 78 breast cancer patients (left panel). Each row represents a tumour and each column a gene, whose name is labelled between b and c. Genes are ordered according to their correlation coefficient with the two prognostic groups. Tumours are ordered by the correlation to the average profile of the good prognosis group (middle panel). Solid line, prognostic classifier with optimal accuracy; dashed line, with optimized sensitivity. Above the dashed line patients have a good prognosis signature, below the dashed line the prognosis signature is poor. The metastasis status for each patient is shown in the right panel: white indicates patients who developed distant metastases within 5 years after the primary diagnosis; black indicates patients who continued to be disease-free for at least 5 years. c, Same as for b, but the expression data matrix is for tumours of 19 additional breast cancer patients using the same 70 optimal prognostic marker genes. Thresholds in the classifier (solid and dashed line) are the same as b. (See Fig. 1 for colour scheme.) Gene Expression of Breast Cancer predicts disease-free outsome (Nature 2002 Friend et al) NOW A CLINICAL ASSAY!!: ONCOTYPE Agilent arrays

3. Deep sequencing Deep or “next generation” sequencing involves three steps 1.Creation of a library (a mixture of DNA that may be a whole genome, or part of a genome from many individuals, or a whole “transcriptome” etc) 2. Partition of the library into single DNA molecules, and amplification of those molecules in situ. 3. Parallel sequencing of all the DNA molecules in a single reaction.

4. Illumina Solexa sequencing http://seqanswers.com/forums/showthread.php?t=21

5. Generation of a single-stranded template DNA library Emulsion-based clonal amplification of the library Data generation via sequencing-by- synthesis Data analysis using different bioinformatics tools 454 sequencing (Roche) SOLiD sequencing (Applied Biosystems)

6. Applications of Next Generation Sequencing Sequence capture protocol: resequence a region hundreds of times Whole genome shotgun sequencing One can use DNA pools to completely describe variation in a region Cut a single genome to bits, sequence the pieces, and use a bioinformatic program to “reassemble” the genome

7. Applications of Next Generation Sequencing ChIP-Seq: Chromatin Immunoprecipitation sequencing. Use an antibody to “pull-down” a DNA binding protein. Sequence the DNA fragments that are attached to the protein.

8. 01_02.jpg CH 3 5-methyl- The “fifth base” 8

9. 5-methyl-C facts Only occurs in “C’s” next to “G’s.” called CpG sites. Is bound specifically by methyl-CpG binding proteins. Acts as a tag for higher order repression of gene promoters. Is a mechanism involved in parental gene imprinting. Unmethylated CpG associated with open chromatin state. CpG’s overrepresented in gene promoters: CpG islands. Closed chromatin, not accessible Open chromatin, accessible to transcription factors 9

10. DNA methylation in development DNA methylation is metastable during development

11. Bisulfite Modification of DNA to Detect Methylated Cytosines DNA sample: --ACTTCGCGACCT-- ( C =methylated) Incubation of DNA with sodium bisulfite and hydroquinone modifies unmethylated cytosine residues to uracil (U). Methylated C’s are protected from this modification. Modified DNA: --AUTTCGCGAUUT-- If all C’s are unprotected (unmethylated), ALL will be modified to U’s: --ACTTCGCGACCT-- becomes --AUTTUGUGAUUT-- after modification

12. What can we do with bisulfite DNA? Sequence it using standard techniques Genotype it using PCR-based and microarrays Large region and whole genome using deep sequencing

13. DNA methylation patterns are specific to each tissue: Bisulfite DNA genotyped on an Illumina array