StructuralVariationintheHumanGenome.pptVIP

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Size Spectrum of Human Genomic Variation Scherer et al. 2007 Types of Structural Variation Hurles et al. 2008 100 101 102 103 104 105 106 107 108 109 BAC-, oligo/SNP array, (FISH) Microscope HR-CGH-arrays Sanger sequencing [bp] 454-PEM [adapted from Lupski et al. Nat Genet 2007] The resolution gap in SV analysis Breakpoint prediction to within PCR range 454-PEM (short-read) 2nd-gen sequencing Paired End Mapping Korbel et al. Science 19 October 2007: Vol. 318. no. 5849, pp. 420 - 426 Mechanism Distribution Published SVs 1KG SVs 2. SV-CapSeq analytical pipeline Map reads using Megablast; Best hit placement Intersect placements with target regions Precisely align reads with Needleman-Wunsch to identify split reads: SV validated, breakpoint sequence found 1. Targeted Sequencing hybridize genomic DNA to capture array wash away unbound fraction Elute off target DNA Sequence with 454 Titanium (~400 bp reads) Array Capture Sequencing Roche-NimbleGen 2000bp 2000bp 2000bp 2000bp Deletion 500bp 500bp Insertion Represented on the capture tiling array (not to scale) Inversion 5000bp 5000bp 5000bp 5000bp SV-CapSeq: Array Design Contents of the SV-CapSeq array v1.0 2.1 million oligomers tiling the target regions of the genome: 1839 deletion CNVs from (mostly) short read Solexa data (1000 Genome Project) From long read 454 paired-end data: 575 deletion CNVs 296 insertions CNVs 191 inversions SVs (plus Split-Read indel predictions, Zhengdong Zhang) Confirmation rate by overlap Reads Zero level Read count Reference Genome Reference Read Deletion 2. Split read 3. Read depth (or aCGH) Mapping Reference Genome Deletion Mapping 1. Paired ends Reference Genome Mapping Reference Sequenced paired-ends Deletion Methods to Find SVs 4. Local Reassembly [Snyde