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Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing

Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing. Rosa Maria Alvarez Massachusetts Institute of Technology Class of 2009. Purpose. Create jumping libraries to detect genomic rearrangements in cancer cells using next generation sequencing technologies.

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Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing

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  1. Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing Rosa Maria Alvarez Massachusetts Institute of Technology Class of 2009

  2. Purpose Create jumping libraries to detect genomic rearrangements in cancer cells using next generation sequencing technologies.

  3. Next Generation Sequencing • Millions of reads in parallel • Reads are short • Structural rearrangements are hard to detect

  4. 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

  5. DNA Methylation Shear DNA Genomic DNA EcoP15 I SAM Ligation of EcoP15 I CAP Adapters Biotinylated Internal Adapters PCR Adapters EcoP15 I Digestion Sinefungin, ATP EcoP15 I Digested Molecule 27bp DNA tag

  6. 2-Log DNA Ladder 10kb 5kb 0.2kb 0.1kb 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 25 cycles 28 cycles 154bp 22 cycles 25 cycles 28 cycles 1. Phusion 5. 0.5uL template 2. 1uL template 6. 1uL template 3. Phusion + SOLiD primers 7. 3uL template 4. 0.2uL template Trial PCR

  7. Amplification and Sequencing • Large Scale PCR amplification of library • Library QC by conventional cloning and Sanger sequencing • Jumping library paired end sequenced by SOLiD

  8. 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

  9. 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;

  10. 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

  11. Acknowledgements • Louise Williams, PhD • Manuel Garber, PhD • Andreas Gnirke, PhD • Shawna Young (Program Coordinator) • Bruce Birren, PhD Thank you for your support!

  12. END

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