Sequence Capture and Targeted Re-sequencing

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Presentation transcript:

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

Sequence Capture / Genome reduction: Ultra deep sequencing

Modifications done to Agilent’s in-solution hybrid capture method Sample QC_ 260/230 and 260/280: 2.0 to 2.2 Fragmentation on Covaris

Covaris fragment profiles observed on DNA 1000 LabChip

End Repair Klenow exo- and ATP

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

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

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

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

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%).

Coverage achieved: Maximum: 202.98x Average: 130.92x Minimum: 50.95x