Detection of Fusion Genes by Targeted Roche 454 Sequencing

Transcription

1 Detection of Fusion Genes by Targeted Roche 454 Sequencing Hans-Ulrich Klein 1, Christoph Bartenhagen 1, Alexander Kohlmann 2, Vera Grossmann 2, Christian Ruckert 1, Torsten Haferlach 2, Martin Dugas 1 1 Department of Medical Informatics and Biomathematics, University of Münster 2 Munich Leukemia Laboratory 55. GMDS Jahrestagung 07 September 2010 Motivation Fusion genes caused by structural variants are an important characteristic for prognosis and therapy of cancer. Study at the Munich Leukemia Laboratory: Evaluation of Roche 454 Sequencing to detect structural variants

2 Motivation Fusion genes caused by structural variants are an important characteristic for prognosis and therapy of cancer. Study at the Munich Leukemia Laboratory: Evaluation of Roche 454 Sequencing to detect structural variants Implement a bioinformatics work flow for the detection of structual variants (the method should not make use of prior knowledge) Assess the reliability of the technology Structural variants

3 Experimental design Targeted Sequencing by capture arrays: A1: RUNX1, CBFB, MLL + exons of 92 other genes (5 samples) A2: MLL (10 samples) A3: RUNX1 (5 samples) A4: PDGFRB (2 samples) Experimental design Targeted Sequencing by capture arrays: A1: RUNX1, CBFB, MLL + exons of 92 other genes (5 samples) A2: MLL (10 samples) A3: RUNX1 (5 samples) A4: PDGFRB (2 samples) reads per sample in median 325bp median read length

4 Analysis work flow 1 Image and signal processing 2 Preprocessing 3 Sequence alignment 4 Filter chimeric reads 5 Detect putative breakpoints 6 Visualization of chromosomal aberrations Sequence alignment Common aligners for 454: SSAHA2, BLAT, BWA-SW We used BWA-SW (H. Li, R. Durbin, Bioinformatics 2010) Only best sequence alignment is reported Alignment is non-overlapping on the query sequence

5 Sequence alignment Common aligners for 454: SSAHA2, BLAT, BWA-SW We used BWA-SW (H. Li, R. Durbin, Bioinformatics 2010) Only best sequence alignment is reported Alignment is non-overlapping on the query sequence Aligned Reads On Target Cvg. Chimeric A (91.6%) 63.0% A (90.8%) 5.1% Filter chimeric reads 1 No more than two local alignments 2 At least one segment must align to the target region 3 No linker sequence between local alignments 4 Local alignments must not be located to close to each other (> 1kb) 5 Remove duplicated reads Chimeric A A

6 Cluster chimeric reads Define distance d between two chimeric reads x, y: d = if chrom. or strand and orientation are not compatible, d = (x A y A ) 2 +(x B y B ) 2 else. Hierarchical clustering, cut dendrogram at d = 100 Compute consensus breakpoint for each cluster Merge breakpoints presumably caused by the same variant Read x Chr A Read y Chr B Cluster chimeric reads Define distance d between two chimeric reads x, y: d = if chrom. or strand and orientation are not compatible, d = (x A y A ) 2 +(x B y B ) 2 else. Hierarchical clustering, cut dendrogram at d = 100 Compute consensus breakpoint for each cluster Merge breakpoints presumably caused by the same variant Read x Read y x A ya x B yb Chr B Chr A

7 Summary detected breakpoints Cluster Size Cvg. Dominant Cluster Bp 1 Bp 2 N01 - A N03 - A N04 - A N05 - A N06 - A N14 - A N16 - A N17 - A N20 - A N21 - A N38 - A N39 - A N40 - A N41 - A N42 - A N27 - A N28 - A N29 - A N30 - A N33 - A N36 - A N37 - A Sample N01 - Visualization (1/2) deletion insertion mismatch breakpoint 67,121,088 67,121,533 5' 3' + MYH11 CBFB + 3' 5' 15,815,687 15,815,191 15,815,189 15,814, ' 3' CBFB MYH11 3' 5' + 67,120,631 67,121,086

8 Sample N01 - Visualization (2/2) CCAGTCCAAAAACCTCCTTCCATTTCCGATGATAGTTCGCTATGAAAAAGTAATCTCCAAATATAATGTAGCTGAAGAGCACTTTTTAGAAAATGATTCC CCAGTCCAAAAACCTCCTTCCATTTCCGATGATAGTTCGCTATGAAAAAGTAATCTCCAAATATAATGTAGCTGAAGAGCACTTTTTAGAAAATGATTCC CCAGTCCAAAAACCTCCTTCCATTTCCGATGATAGTTCGCTATGAAAAAGTAATCTCCAAATATAATGTAGCTGAAGAGCACTTTTTAGAAAATGATTCC + 5' CCAGTCCAAAAACCTCCTTCCATTTCCGATGATAGTTCGCTATGAAAAAGTAATCTCCAAATATAATGTAGCTGAAGAGCACTTTTTAGAAAATGATTCC 3' MYH11 CBFB 3' GGTCAGGTTTTTGGAGGAAGGTAAAGGCTACTATCAAGCGATACTTTTTCATTAGAGGTTTATATTACATCGACTTCTCGTGAAAAATCTTTTACTAAGG 5' + GGTCAGGTTTTTGGAGGAAGGTAAAGGCTACTATCAAGCGATACTTTTTCATTAGAGGTTTATATTACATCGACTTCTCGTGAAAAATCTTTTACTAAGG GCAAAATACATACAAAAGCTTTCAACAGTTGTTCCATTAATTGTCAAATAGCCAGGAGCTAGCCTCGCATGGACTGGTGAATAGCACAGAGGGTGGGCAG GCAAGATACATACAAAAGCTTTCAACAGTTGTTCCATTAATTGTCAAATAGCCAGGAGCTAGCCTCGCATGGACTGGTGAATAGCACAGAGGGTGGGCAG GCAAAATACATACAAAAGCTTTCAACAGTTGTTCCATTAATTGTCAAATAGCCAGGAGCTAGCCTCGCATGGACTGGTGAATAGCACAGAGGGTGGGCAG ATACAAAAGCTTTCAACAGTTGTTCCATTAATTGTCAAATAGCCAGGAGCTAGCCTCGCATGGACTGGTGAATAGCACAGAGGGTGGGCAG 5' GCAAAATACATACAAAAGCTTTCAACAGTTGTTCCATTAATTGTCAAATAGCCAGGAGCTAGCCTCGCATGGACTGGTGAATAGCACAGAGGGTGGGCAG 3' + CBFB MYH11 + 3' CGTTTTATGTATGTTTTCGAAAGTTGTCAACAAGGTAATTAACAGTTTATCGGTCCTCGATCGGAGCGTACCTGACCACTTATCGTGTCTCCCACCCGTC 5' CGTTTTATGCATGTTTTCGAAAGTTGTCAACAAGGTAATTAACAGTTTATCGGTCCTCGATCGGAGCGTACCTGACCACTTACCGTGTCTCCCACCCCTC CGTTTTATGTATGTTTTCGAAAGTTGTCAACAAGGTAATTAACAGTTTATCGGTCCTCGATCGGAGCGTACCTGACCACTTATCGTGTCTCCCACCCGTC CGTTTTATGTATGTTTTCGAAAGTTGTCAACAAGGTAATTAACAGTTTATCGGTCCTCGATCGGAGCGTACCTGACCACTTATCGTGTCTCCCACCCGTC CGTTTTATGTATGTTTTCGAAAGTTGTCAACAAGGTAATTAACAGTTTATCGGTCCTCGATCGGAGCGTACCTGACCACTTATCGTGTCTCCCACCCGTC Conclusions Unsupervised approach to filter out interesting reads and to propose a few putative structural variants One single chimeric read is not sufficient coverage Implemented as R-package R453Plus1Toolbox Future: Include biological knowledge about common variants and improve merging

9 Conclusions Unsupervised approach to filter out interesting reads and to propose a few putative structural variants One single chimeric read is not sufficient coverage Implemented as R-package R453Plus1Toolbox Future: Include biological knowledge about common variants and improve merging Thank You!