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Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

Murphy SJ, Cheville JC, Zarei S, Johnson SH, Sikkink RA, Kosari F, Feldman AL, Eckloff BW, Karnes RJ, Vasmatzis G - DNA Res. (2012)

Bottom Line: Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols.Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation.Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Medical Sciences Building 2, 200 First St., SW, Rochester, MN 55905, USA. murphy.stephen@mayo.edu

ABSTRACT
High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

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Related in: MedlinePlus

Validation of chromosome translocation events: mapping of two independent translocation events are depicted; a balanced translocation between chromosomes 1 and 12 (a–c) and a complex rearrangement between chromosomes 8 and 11 (e and f). (a) MP sequences mapping to chromosomes 1 and 12 for GP3 and GP4 are depicted above and below the zero axis, respectively, as red or blue dots dependent on the direction of the sequence read mapping to the reference genome. Horizontally linked red and blue dots closest to this axis depict normal mapping MP sequences to chromosome 1 or 12 alone. MP sequences linked vertically depict translocations between these two chromosomes. (b) PCR validation using primers specific to the t(1-12) event (i) for GP3 and GP4 (lanes 5 and 6) and the t(12-1) event (ii) for GP3 and GP4 (lanes 9 and 10). gDNA (lanes 2 and 8) and different prostate tumour tissues (PR1 and PR2, lanes 3 and 4) were used as controls together with 1 kb ladder (lanes 1 and 7). (c) Schematic representation of predicted VASH2 and AK055062 fusion products resulting from the translocation events described. (d) Mapping of MP sequences to three inter-linked events at genomic loci 8q22.2b, 11q13.2c and 11q24.2b. Events 1 and 2 describe two t(8-11) translocation events linking VPS13B to CHKA (i) and CHEK1 (ii), respectively. Event 3 describes an r(11-11) intra-chromosomal rearrangement linking CHEK1 with SASP3 (iii). (e) PCR validation 1% agarose gels are presented for four different primer sets (1–4) for event 1 (i), five different primers sets (1–5) for event 2 (ii) and two primer sets (1 and 2) for event 3 (iii) for the GP3 and GP4 WGA DNA and gDNA as control. (f) Schematic representation of the three fusion events on chromosome 8 (red) and 11 (blue), describing the potential products and the impact on the gene regions involved. Arrows describe the coding direction of the genes and an X represents the loss of the promoter regions.
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DSS021F3: Validation of chromosome translocation events: mapping of two independent translocation events are depicted; a balanced translocation between chromosomes 1 and 12 (a–c) and a complex rearrangement between chromosomes 8 and 11 (e and f). (a) MP sequences mapping to chromosomes 1 and 12 for GP3 and GP4 are depicted above and below the zero axis, respectively, as red or blue dots dependent on the direction of the sequence read mapping to the reference genome. Horizontally linked red and blue dots closest to this axis depict normal mapping MP sequences to chromosome 1 or 12 alone. MP sequences linked vertically depict translocations between these two chromosomes. (b) PCR validation using primers specific to the t(1-12) event (i) for GP3 and GP4 (lanes 5 and 6) and the t(12-1) event (ii) for GP3 and GP4 (lanes 9 and 10). gDNA (lanes 2 and 8) and different prostate tumour tissues (PR1 and PR2, lanes 3 and 4) were used as controls together with 1 kb ladder (lanes 1 and 7). (c) Schematic representation of predicted VASH2 and AK055062 fusion products resulting from the translocation events described. (d) Mapping of MP sequences to three inter-linked events at genomic loci 8q22.2b, 11q13.2c and 11q24.2b. Events 1 and 2 describe two t(8-11) translocation events linking VPS13B to CHKA (i) and CHEK1 (ii), respectively. Event 3 describes an r(11-11) intra-chromosomal rearrangement linking CHEK1 with SASP3 (iii). (e) PCR validation 1% agarose gels are presented for four different primer sets (1–4) for event 1 (i), five different primers sets (1–5) for event 2 (ii) and two primer sets (1 and 2) for event 3 (iii) for the GP3 and GP4 WGA DNA and gDNA as control. (f) Schematic representation of the three fusion events on chromosome 8 (red) and 11 (blue), describing the potential products and the impact on the gene regions involved. Arrows describe the coding direction of the genes and an X represents the loss of the promoter regions.

Mentions: Figure 3a–c presents the validation of a balanced translocation event between 1q32.3c and 12q21.33a, involving genes VASH2 and AK055062, respectively (Table 1). The GP3 and GP4 cell populations recorded 11 and 21 MP reads, respectively (Table 1), which are mapped in Fig. 3a. The upper and lower parts of the figures refer to chromosomes 1 and 12, respectively. MP reads are illustrated as either blue or red dots, depending on the polarity of the MP sequence mapping to the reference genome, with a line linking the paired reads. Central horizontally linked red-to-blue dots represent intra-chromosomal mapping MP fragments with the concordant bridged coverage spans (3–5 kb). Vertically linked red-to-blue dots are associated with MPs specific to this translocation event between chromosomes 1 and 12 (Fig. 3a, i and ii). Two sets of red/blue-linked MP events spanning the two chromosomes are observed, red-to-blue and blue-to-red. This is indicative of a balanced translocation event between the two chromosomes, where genomic regions have been exchanged between the two chromosomes resulting in t(1-12) and t(12-1) translocation products (Fig. 3c). The regions where the red–blue and blue–red mapping events converge indicate the location of the translocation breakpoints.Figure 3.


Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

Murphy SJ, Cheville JC, Zarei S, Johnson SH, Sikkink RA, Kosari F, Feldman AL, Eckloff BW, Karnes RJ, Vasmatzis G - DNA Res. (2012)

Validation of chromosome translocation events: mapping of two independent translocation events are depicted; a balanced translocation between chromosomes 1 and 12 (a–c) and a complex rearrangement between chromosomes 8 and 11 (e and f). (a) MP sequences mapping to chromosomes 1 and 12 for GP3 and GP4 are depicted above and below the zero axis, respectively, as red or blue dots dependent on the direction of the sequence read mapping to the reference genome. Horizontally linked red and blue dots closest to this axis depict normal mapping MP sequences to chromosome 1 or 12 alone. MP sequences linked vertically depict translocations between these two chromosomes. (b) PCR validation using primers specific to the t(1-12) event (i) for GP3 and GP4 (lanes 5 and 6) and the t(12-1) event (ii) for GP3 and GP4 (lanes 9 and 10). gDNA (lanes 2 and 8) and different prostate tumour tissues (PR1 and PR2, lanes 3 and 4) were used as controls together with 1 kb ladder (lanes 1 and 7). (c) Schematic representation of predicted VASH2 and AK055062 fusion products resulting from the translocation events described. (d) Mapping of MP sequences to three inter-linked events at genomic loci 8q22.2b, 11q13.2c and 11q24.2b. Events 1 and 2 describe two t(8-11) translocation events linking VPS13B to CHKA (i) and CHEK1 (ii), respectively. Event 3 describes an r(11-11) intra-chromosomal rearrangement linking CHEK1 with SASP3 (iii). (e) PCR validation 1% agarose gels are presented for four different primer sets (1–4) for event 1 (i), five different primers sets (1–5) for event 2 (ii) and two primer sets (1 and 2) for event 3 (iii) for the GP3 and GP4 WGA DNA and gDNA as control. (f) Schematic representation of the three fusion events on chromosome 8 (red) and 11 (blue), describing the potential products and the impact on the gene regions involved. Arrows describe the coding direction of the genes and an X represents the loss of the promoter regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DSS021F3: Validation of chromosome translocation events: mapping of two independent translocation events are depicted; a balanced translocation between chromosomes 1 and 12 (a–c) and a complex rearrangement between chromosomes 8 and 11 (e and f). (a) MP sequences mapping to chromosomes 1 and 12 for GP3 and GP4 are depicted above and below the zero axis, respectively, as red or blue dots dependent on the direction of the sequence read mapping to the reference genome. Horizontally linked red and blue dots closest to this axis depict normal mapping MP sequences to chromosome 1 or 12 alone. MP sequences linked vertically depict translocations between these two chromosomes. (b) PCR validation using primers specific to the t(1-12) event (i) for GP3 and GP4 (lanes 5 and 6) and the t(12-1) event (ii) for GP3 and GP4 (lanes 9 and 10). gDNA (lanes 2 and 8) and different prostate tumour tissues (PR1 and PR2, lanes 3 and 4) were used as controls together with 1 kb ladder (lanes 1 and 7). (c) Schematic representation of predicted VASH2 and AK055062 fusion products resulting from the translocation events described. (d) Mapping of MP sequences to three inter-linked events at genomic loci 8q22.2b, 11q13.2c and 11q24.2b. Events 1 and 2 describe two t(8-11) translocation events linking VPS13B to CHKA (i) and CHEK1 (ii), respectively. Event 3 describes an r(11-11) intra-chromosomal rearrangement linking CHEK1 with SASP3 (iii). (e) PCR validation 1% agarose gels are presented for four different primer sets (1–4) for event 1 (i), five different primers sets (1–5) for event 2 (ii) and two primer sets (1 and 2) for event 3 (iii) for the GP3 and GP4 WGA DNA and gDNA as control. (f) Schematic representation of the three fusion events on chromosome 8 (red) and 11 (blue), describing the potential products and the impact on the gene regions involved. Arrows describe the coding direction of the genes and an X represents the loss of the promoter regions.
Mentions: Figure 3a–c presents the validation of a balanced translocation event between 1q32.3c and 12q21.33a, involving genes VASH2 and AK055062, respectively (Table 1). The GP3 and GP4 cell populations recorded 11 and 21 MP reads, respectively (Table 1), which are mapped in Fig. 3a. The upper and lower parts of the figures refer to chromosomes 1 and 12, respectively. MP reads are illustrated as either blue or red dots, depending on the polarity of the MP sequence mapping to the reference genome, with a line linking the paired reads. Central horizontally linked red-to-blue dots represent intra-chromosomal mapping MP fragments with the concordant bridged coverage spans (3–5 kb). Vertically linked red-to-blue dots are associated with MPs specific to this translocation event between chromosomes 1 and 12 (Fig. 3a, i and ii). Two sets of red/blue-linked MP events spanning the two chromosomes are observed, red-to-blue and blue-to-red. This is indicative of a balanced translocation event between the two chromosomes, where genomic regions have been exchanged between the two chromosomes resulting in t(1-12) and t(12-1) translocation products (Fig. 3c). The regions where the red–blue and blue–red mapping events converge indicate the location of the translocation breakpoints.Figure 3.

Bottom Line: Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols.Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation.Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Medical Sciences Building 2, 200 First St., SW, Rochester, MN 55905, USA. murphy.stephen@mayo.edu

ABSTRACT
High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

Show MeSH
Related in: MedlinePlus