Sequence Capture and Targeted Re-sequencing Thahira Rahman, Institute of Human Genetics, Newcastle...
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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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Transcript of Sequence Capture and Targeted Re-sequencing Thahira Rahman, Institute of Human Genetics, Newcastle...
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:
