Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing
Rosa Maria AlvarezMassachusetts Institute of Technology
Class of 2009
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Purpose
Create jumping libraries to detect
genomic rearrangements in
cancer cells using next generation
sequencing technologies.
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Millions of reads in parallel Reads are short Structural rearrangements are
hard to detect
Next Generation Sequencing
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Why create jumping libraries?
Paired end reads detect translocations Jumping libraries combine two sequences that
are far apart on a small sequenceable construct
Pairs at least a few kb apart are needed to scan a human genome
Example: 10 million paired reads, 3 kb apart provides 10x coverage
EcoP15 I Digestion Sinefungin, ATP
DNA Methylation
EcoP15 I
SAM
Ligation of EcoP15 I
CAP Adapters
Biotinylated Internal
Adapters
Genomic DNA
Shear DNA
EcoP15 I Digested Molecule 27bp DNA tag
PCR Adapte
rs
154bp
22 cycles
25 cycles 28 cycles
1 2 3 4 5 6 7
1. Phusion 5. 0.5uL template2. 1uL template 6. 1uL template3. Phusion + SOLiD primers 7. 3uL template4. 0.2uL template
2-Log DNA Ladder
10kb 5kb
0.2kb
0.1kb
1 2 3 4 5 6 7 1 2 3 4 5 6 7
25 cycles 28 cycles
Trial PCR
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Amplification and Sequencing
Large Scale PCR amplification of library Library QC by conventional cloning and
Sanger sequencing Jumping library paired end sequenced
by SOLiD
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Sanger Sequencing of Test Library; Jump Size
Number of clones sequenced: 384
Number of aligning clones: 375
Number of aligning clones with both ends :148
Number of chimeras: 43
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SOLiD Sequencing Results
13 million paired end reads generated Total aligning reads 813444
2% aligned correctly 27% chimeras
Good news: We did find the translocation! BCR-ABL (9,22) translocation Philadelphia Chromosome 963_1200_515_3 chr9: 132597006-132597031;
chr22: 21960248-21960273;
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Conclusions
Constructed jumping library Structure of library molecules verified by
Sanger sequencing Jump size within the expected range
Library was sequenceable by SOLiD First mammalian SOLiD sequencing runs at the
Broad Successfully detected the Philadelphia
Chromosome translocation
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Acknowledgements
Louise Williams, PhD Manuel Garber, PhD Andreas Gnirke, PhD
Shawna Young (Program Coordinator) Bruce Birren, PhD
Thank you for your support!
END