Sequence Capture and Targeted Re-sequencing

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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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Sequence Capture / Genome reduction
Ultra deep sequencing
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Modifications done to Agilents in-solution
hybrid capture method
(5µg)
Sample QC_ 260/230 and 260/280 2.0 to 2.2
Fragmentation on Covaris
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Covaris fragment profiles observed on DNA 1000
LabChip
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End Repair
Klenow exo- and ATP
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Ligate Multiplex PE adapters
SPRI bead purification
Ligated samples checked on DNA 1000 LabChip
(please refer previous slides)
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Probes designed on eArray
Custom synthesised Blocks used for Hyb
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Post hybridisation PCR involve indexing of
libraries
Quantification of adapted and enriched libraries
on High Sensitivity LabChip
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Equimolar pooling of libraries
50bp Multiplex PE Illumina sequencing
Data Analyses
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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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Coverage achieved
Maximum 202.98x Average 130.92x Minimum 50.95x