Analysis of complex genomic and epigenomic variation in humans using advanced target-capture sequencing approaches
Alexander E. Urban
Wednesday, October 14, 2015
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Chapters
Introduction
Outline of talk
CLIP-Cap: Combined Long-Insert Paired-End and Capture Sequencing
Circularization based, Long-Insert, Paired-End sequencing
CLIP-Cap
Original detection of the 22q13 aberration with MLPA-PCR
Reanalysis with SNP array and ddPCR: signs of a more complex situation
Growing multiple clonal subcultures and ddPCR: multiple genotypes indicate mosaicism
FISH confirms complex rearrangements instead of simple deletion
Karyotype confirms mosaic presence of isodicentric chromosome 22
Multiple approaches combined yield model of complex mosaicism
Combined Long-Insert Paired-End and Capture Sequencing
We ordered a NimbleGen
Nextera Mate Pair library preparation
Roche NimbleGen SeqCap EZ chr22 capture
Mapping the sequencing reads shows very good on-target rates
CLIP-Cap captured PE-reads detect the isodicentric junctions with high accuracy
CLIP-Cap detects the isodicentric junctions with high accuracy in a single experiment
next: apply CLIP-Cap to other samples with complex rearrangements
Step 1: Nextera Mate-Pair libraries
Philadelphia chromosome
K562: first CML cell line established in 1970
Results: all capture samples – whole genome view
The three main Philadelphia junction points are detected and resolved by CLIP-Cap
SUP-B15
Kasumi-4: three-way translocation
Kasumi-4
K562
K562 chr22
The captured PE reads pile up
For precise and comprehensive analysis of complex genomic rearrangements
2. Validation of somatic
Components of the Human Genome
Detection of somatic retro-transposition in human brain pt. 1
First round of project: two individuals, three tissues, DNA from unsorted cells
Detection of somatic retro-transposition in human brain pt. 2
First round of project: two individuals, three tissues, DNA from unsorted cells
Calling novel transposon insertions using Capture-sequencing data
20,000 targets for custom capture re-sequencing (high- and low-confidence) pt 2.
Calling novel transposon insertions using Capture-sequencing data
High consistency of TE calls between whole-genome and capture sequencing
3. SeqCap Epi: target capture for DNA methylation analysis
NimbleGen SeqCap Epi Workflow
CpGiant for DNA methylation analysis
We are using CpGiant on several projects
CpGiant read alignments – some examples
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