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
(5µg)
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)
Probes designed on eArray
Adapter linked gDNA fragments
SureSelect Biotinylated RNA Baits
Hybridisation (@ 65C for 24h)
Streptavidin coated
magnetic beads
Custom
synthesised Blocks
used for Hyb
Hybrid capture using MPC
Unbound fragments
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