Sequence Capture and Targeted Re-sequencing

Thahira Rahman, Institute of Human Genetics, Newcastle University,

Newcastle upon Tyne

Ultra deep sequencing

Sequence Capture / Genome reduction:

Modifications done to Agilent’s in-solution hybrid capture method

Fragmentation on Covaris

Sample QC_ 260/230 and 260/280: 2.0 to 2.2

(5µg)

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)

Probes designed on eArray

Adapter linked gDNA fragments SureSelect Biotinylated RNA Baits

Hybridisation (@ 65C for 24h)

Streptavidin coated magnetic beads

Hybrid capture using MPCUnbound 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%).

Maximum: 202.98xAverage: 130.92xMinimum: 50.95x

Coverage achieved: