Detection of Genomic Rearrangements in K562 cells using Paired End Sequencing

Rosa Maria AlvarezMassachusetts Institute of Technology

Class of 2009

2

Purpose

Create jumping libraries to detect

genomic rearrangements in

cancer cells using next generation

sequencing technologies.

3

Millions of reads in parallel Reads are short Structural rearrangements are

hard to detect

Next Generation Sequencing

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

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

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!

END