CpG (Whole genome bisulfite sequencing; WGBS) MeDip-Seq 1 DNA CpG-rich 1. SureSelect Reduced representation bisulfite sequencing (RRBS) DNA CpG PCR DN

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1 Agilent SureSelect XT Human Methyl-Seq 1 DNA Agilent SureSelect XT Human Methyl-Seq 1 SureSelect DNA Agilent SureSelect XT Human Methyl-Seq 370 CpG Human Methyl- Seq CpG shore shelf CpG 4000 CpG 2kb CpG shore SureSelect XT Human Methyl-Seq DNA (differentially methylated regions; DMRs)

2 CpG (Whole genome bisulfite sequencing; WGBS) MeDip-Seq 1 DNA CpG-rich 1. SureSelect Reduced representation bisulfite sequencing (RRBS) DNA CpG PCR DNA CpG-rich SureSelect XT Human Methyl-Seq 1 DNA SureSelect Target Enrichment System SureSelect SureSelect XT Human Methyl- Seq CpG Gencode CpG shore shelf DNase I Refseq Ensembl regulatory features DMRs 84 Mb 370 CpG CpG DMR Gencode DMR regulatory features CpG shore shelf 4 kb DNase I Refseq Ensembl regulatory features 2

3 SureSelect XT Human Methyl-Seq (IMR 90) (R > 0.93) 2 (>10 ) IMR90 SureSelect XT Human Methyl-Seq (whole genome sequencing; WGS) 2 2. SureSelect XT Human Methyl-Seq WGS (R > 0.93) SureSelect HCT116 (DNMT1-/- DNMT3b-/-) DNA 3 HCT116 DNA DNA Agilent SureSelect XT Human Methyl-Seq 4Gbp 91Gbp CpG CpG ( 4) 3. HCT116 (A) (B) CpGs covered (%) A Whole-genome sequencing (91 Gbp) SureSelect XT Human Methyl-Seq (4Gbp) 1X 5X 10X 15X 20X 25X 30X Coverage of at least... CpGs covered (%) B Whole-genome sequencing (91 Gbp) SureSelect XT Human Methyl-Seq (4Gbp) 1X 5X 10X 15X 20X 25X 30X Coverage of at least (180 Gb 91 Gb ) SureSelect XT Human Methyl-Seq (10 Gb 4 Gb ) 370 CpG (A) CpG 190 CpG (B) %CpG 3

4 SureSelect XT Human Methyl-Seq 5 DMRs 200 bp 208, , DMR DMRs SureSelect XT Human Methyl-Seq DMRs DMRs 1 comprehensive high-throughput arrays for relative methylation (CHARM) HOXA3 DMRs ( 52 % 72 %) Methyl-Seq SureSelect XT Human Methyl-Seq HOXA3 HOXA3/HOXA4 DMR ( 6) 5. SureSelect XT Human Mehtyl-Seq 5 6. ( ) ( ) DMR DMR 1 4

5 SureSelect XT Human Methyl-Seq DNA 1 DNA (bisulfite treated: BST) DNA demultiplex Agilent SureSelect XT Methyl- Seq duplicate % ( 7) 1. DNA (BST) BST DNA BST DNA BST BST Bismark ( 8) C T G A ( C-T ) Bismark C T G A Bismark strand Alignment Genomic fragment Sequence after bisulfite treatment me me ccggcatgtttaaacgct TTGGCATGTTTAAACGTT Duplicate Removal C-to-T G-to-A Read conversion Normalization Detection TTGGTATGTTTAAATGTT (1) (2) ttggtatgtttaaatgtt aaccatacaaatttacaa Forward strand C-to-T converted genome (3) (4) TTAACATATTTAAACATT Align to bisulfite converted genomes ccaacatatttaaacact ggttgtataaatttgtga Forward strand G-to-A converted genome % Methylation Computation Methylation Identification 7. SureSelect XT Methyl-Seq 8. Bismark 1 5 (1) (2) (3) (4) Determine unique best alignment Read all four alignment outputs simultaneously to determine if the sequence can be mapped uniquely

6 2. duplicate PCR duplicate Strand 1 1 ( 9) BST Bismark ( 10) BST Strand C-T ( G-A ) 5. % Bismark % (% m) Bismark Primary Output % ( 11) 9. Duplication : PCR duplicate BS-read corresponds to converted original top strand me me 5 TTGGCATGTTTAAACGTT 3 5 ccggcatgtttaaacgct 3 xz..h...z.h Bismark Bismark ( 1) Bisulfite read genomic sequence Methylation call n z Z x X h H Reference GTT GTT GTT 11. unmethylated C in CpG context methylated C in CpG context unmethylated C in CHG context methylated C in CHG context unmethylated C in CHH context methylated C in CHH context k %m = k n 6

7 Agilent SureSelect XT Human Methyl-Seq SureSelect XT Human Methyl-Seq DNA DNA 1 SureSelect XT Human Methyl-Seq SureSelect XT Human Methyl-Seq DMRs DMR 1. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq Applications. Bioinformatics, 2011, 27: Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic difference. Nature, 2009, 462:

8 : * Genome Preparation : ( 1 ) USAGE: bismark_genome_preparation [options] <path_to_genome_folder> Sample command : bismark_genome_preparation --path_ to_bowtie /usr/local/bowtie/ -- verbose/data/genomes/homo_sapiens /GRCh37/ % methylation computation The genome_methylation_ bismark2bedgraph_v3.pl ac.uk/projects/download.html#bismark * Bismark Bismark Alignment : USAGE: bismark [options] <genome_folder> {-1 <mates1> -2 <mates2> <singles>} 40 bp bismark -q --phred64-quals -n 1 -l 40 --directional /data/genomes/homo_ sapiens/grch37/ s_1_sequence.txt duplicate The deduplicate_bismark_alignment_ output.pl Detection USAGE:./methylation_extractor [options] <fi lenames> methylation_extractor -p --merge_non_ CpG --comprehensive s_1_sequence.txt _bismark_pe.txt Agilent Technologies, Inc., 2012 Published in Japan, August 31, JAJP

Genome-Wide Genetic Analysis More flexibility. More content. CGH CGH CGH CGH Comparative Genomic Hybridization CGH2 DNA KaryotypingFISH CGH BACBacteri

LOH/UPD Genome-Wide Genetic Analysis More flexibility. More content. CGH CGH CGH CGH Comparative Genomic Hybridization CGH2 DNA KaryotypingFISH CGH BACBacterial Artificial Chromosome acgh 60 mer 0.06 kb*