? ? Individual 1Individual 2 1

Questions This is a pedigree for a disease involving a mutation within an imprinted gene. The disease manifests only when the gene is expressed. 1)What is the probability that Individual 1 has the disease? a)0% b)25% c)50% d)75% e)100% 2)What is the probability that Individual 2 has the disease? a)0% b)25% c)50% d)75% e)100% 2

1)The specificity of ChIP-Seq (i.e. which DNA ends up getting sequenced) is caused by a)Formaldehyde treatment b)DNA bending c)DNA/histone affinity d)Antibody pull-down e)Bioinformatic alignment to the genome 2)Most histone modifications were identified using the following feature of mass-spectrometry: a)An increase in relative abundance of the modified peptide b)Fewer peaks on the MS spectrum c)A shift in a peak on the MS spectrum d)Differential digestion of the original protein e)Using a series of different peptidases and comparing MS spectra 3

Science Bomb 4

10 weeks vs. 15, less invasive $494 million valuation 5

Epigenetics, UCSC Browser, NextGen Sequencing Methods Sept. 23,

Illumina HiSeq 2000 NextGen Sequencing 7

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ChIP-Seq is designed for detecting protein-DNA interactions 9

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Consistent Trends in Effects on Gene Expression Repression of gene expression Activation of gene expression 13 DNA Methylation = repression Heterochromatin = repression

UCSC Genome Browser 14 CDS Start Exon Intron 5’UTR 3’UTR CDS End mRNA = transcript after splicing (i.e. just the exons)

UCSC Genome Browser 15 CDS End 3’UTR CDS Start 5’UTR

Applications enabled by high-throughput sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 16

ChIP-Seq mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 17

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 18

Genomic DNA Sequencing Millions of reads Whole Genome AssemblyCopy Number Variation

20 Genome Rearrangements Insertion/Deletion (Indel) Single Nucleotide Variant (SNV) (SNP/mutation) Gene Discovery -start/stop codons -translation is in frame -splice sites -conservation of exons

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 21

Targeted Sequencing: PCR mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 22

23 PCR

24 PCR

Targeted Sequencing: PCR Millions of reads SNV/Indel Discovery

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 26

Targeted Sequencing: Captured mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 27

28 Targeted Sequencing: Captured

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 29

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 30

31 Bisulfite Sequencing ACGTGACGT ATGTGACGT me AUGTGACGT me bisulfite PCR

Genomic DNA Sequencing mRNA Total RNA miRNA Genomic DNA Captured/PCR/IP DNA RNA Whole Genome Assembly Copy Number Variation Genome rearrangements Chromatin structure Regulation of Gene Expression Splice Site Monitoring/Discovery SNV/Indel Identification Gene Discovery Any Species Bisulfite treated Expression Profiling 32