Limits...
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.

Show MeSH

Related in: MedlinePlus

Additional validations in the GP3 and GP4 cancers: (a) 1% agarose gels of PCR validation for 12 additional chromosomal rearrangement events in the GP3 and GP4 cancers, involving two primer sets and using gDNA as control. (b) PCR evaluation of aN and dN tissues for 17 previously validated events in GP3 and GP4 tumours of the same patient. (c) PCR validation in amplified (WGA) and unamplified (nonWGA) GP3 and GP4 tissues for four validated events.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3473372&req=5

DSS021F4: Additional validations in the GP3 and GP4 cancers: (a) 1% agarose gels of PCR validation for 12 additional chromosomal rearrangement events in the GP3 and GP4 cancers, involving two primer sets and using gDNA as control. (b) PCR evaluation of aN and dN tissues for 17 previously validated events in GP3 and GP4 tumours of the same patient. (c) PCR validation in amplified (WGA) and unamplified (nonWGA) GP3 and GP4 tissues for four validated events.

Mentions: In order to further support this novel experimental methodology and bioinformatics algorithm, 12 additional events from Table 1 were selected for PCR validation. All 12 events generated unique PCR products in the prostate cancer that were absent in control gDNA (Fig. 4a). Significantly, all events but one validated in both the GP3 and GP4 samples; however, the intensity of the bands often varied between GP3 and GP4, indicating this did not result from LCM contamination. For several events, the intensity of the band was greater in the GP4 than the GP3, indicating a greater prevalence of the rearrangement in the GP4 cell population. Only the event t(2-23) validated predominantly in GP3 with no or potential very low levels observed in the GP4 (Fig. 4a–c). While it is difficult to confidently define the levels of each genomic aberration in these tumours considering the input of WGA DNA and the semi-quantitative PCR, the band intensities correlated to some extent with the numbers of associated MP reads (Table 1). Sanger sequencing of the validating PCR bands revealed the fusion breakpoints for each event (Supplementary Table S1). Significantly, for each PCR band, the exact fusion junction was identified in both the GP3 and GP4 samples. These data present strong evidence for the clonality of GP3 and GP4 in this prostate cancer. Additionally, the presence of the exact junction breakpoints in both GP3 and GP4 makes these fusion events unlikely to be artefacts of WGA.Figure 4.


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)

Additional validations in the GP3 and GP4 cancers: (a) 1% agarose gels of PCR validation for 12 additional chromosomal rearrangement events in the GP3 and GP4 cancers, involving two primer sets and using gDNA as control. (b) PCR evaluation of aN and dN tissues for 17 previously validated events in GP3 and GP4 tumours of the same patient. (c) PCR validation in amplified (WGA) and unamplified (nonWGA) GP3 and GP4 tissues for four validated events.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473372&req=5

DSS021F4: Additional validations in the GP3 and GP4 cancers: (a) 1% agarose gels of PCR validation for 12 additional chromosomal rearrangement events in the GP3 and GP4 cancers, involving two primer sets and using gDNA as control. (b) PCR evaluation of aN and dN tissues for 17 previously validated events in GP3 and GP4 tumours of the same patient. (c) PCR validation in amplified (WGA) and unamplified (nonWGA) GP3 and GP4 tissues for four validated events.
Mentions: In order to further support this novel experimental methodology and bioinformatics algorithm, 12 additional events from Table 1 were selected for PCR validation. All 12 events generated unique PCR products in the prostate cancer that were absent in control gDNA (Fig. 4a). Significantly, all events but one validated in both the GP3 and GP4 samples; however, the intensity of the bands often varied between GP3 and GP4, indicating this did not result from LCM contamination. For several events, the intensity of the band was greater in the GP4 than the GP3, indicating a greater prevalence of the rearrangement in the GP4 cell population. Only the event t(2-23) validated predominantly in GP3 with no or potential very low levels observed in the GP4 (Fig. 4a–c). While it is difficult to confidently define the levels of each genomic aberration in these tumours considering the input of WGA DNA and the semi-quantitative PCR, the band intensities correlated to some extent with the numbers of associated MP reads (Table 1). Sanger sequencing of the validating PCR bands revealed the fusion breakpoints for each event (Supplementary Table S1). Significantly, for each PCR band, the exact fusion junction was identified in both the GP3 and GP4 samples. These data present strong evidence for the clonality of GP3 and GP4 in this prostate cancer. Additionally, the presence of the exact junction breakpoints in both GP3 and GP4 makes these fusion events unlikely to be artefacts of WGA.Figure 4.

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