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Sequence Capture and Targeted Re-sequencing Thahira Rahman, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne.

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Presentation on theme: "Sequence Capture and Targeted Re-sequencing Thahira Rahman, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne."— Presentation transcript:

1 Sequence Capture and Targeted Re-sequencing Thahira Rahman, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne

2 Ultra deep sequencing Sequence Capture / Genome reduction:

3 Modifications done to Agilents in-solution hybrid capture method Fragmentation on Covaris Sample QC_ 260/230 and 260/280: 2.0 to 2.2 (5µg)

4 Covaris fragment profiles observed on DNA 1000 LabChip

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8 End Repair Klenow exo - and ATP

9 Ligate Multiplex PE adapters SPRI bead purification Ligated samples checked on DNA 1000 LabChip (please refer previous slides)

10 Probes designed on eArray Adapter linked gDNA fragmentsSureSelect Biotinylated RNA Baits Hybridisation 65C for 24h) Streptavidin coated magnetic beads Hybrid capture using MPC Unbound fragments Custom synthesised Blocks used for Hyb

11 Post hybridisation PCR involve indexing of libraries Quantification of adapted and enriched libraries on High Sensitivity LabChip

12 Equimolar pooling of libraries 50bp Multiplex PE Illumina sequencing Data Analyses

13 The data was aligned with hg18 genome build using bowtie Size of the target region covered by SureSelect Human X-Chromosome baits is 3,053,381 bp. Total length of the captured sequence is 3,025,546 bp (99.09%).

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15 Maximum: x Average: x Minimum: 50.95x Coverage achieved:


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