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DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues.

Craig JM, Vena N, Ramkissoon S, Idbaih A, Fouse SD, Ozek M, Sav A, Hill DA, Margraf LR, Eberhart CG, Kieran MW, Norden AD, Wen PY, Loda M, Santagata S, Ligon KL, Ligon AH - PLoS ONE (2012)

Bottom Line: While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations.Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues.Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

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Determination of optimal size among matched DNA fragment size distributions.(A,C,E,G) Agarose gel electrophoresis of reference gDNA (Promega) aliquots after various heat fragmentation times shown adjacent to ImageJ gel analysis of same lanes, molecular weight indicated in bp. Mode fragment size of each smear, as measured with ImageJ, indicated in blue. (B,D,F,H) Agilent 180 K array results of self-hybridizations using reference gDNA (left) and characterized by matching fragment size distributions (B;250/250, D;315/315, F;400/400, H;525/525). Log2 ratios for signal intensities of differentially labeled aliquots (cy5/cy3) are plotted for probes corresponding to chromosome 1 (green;log2ratio<−0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). Data quality was assessed by dLRsd. (I) Mean dLRsd of duplicate (n = 5) or triplicate (n = 2) size-matched self-hybridizations representing seven fragment size distributions plotted by mode fragment length (225, 250, 315, 400, 525, 625, and 680 bp). Error bars indicate SEM.
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pone-0038881-g002: Determination of optimal size among matched DNA fragment size distributions.(A,C,E,G) Agarose gel electrophoresis of reference gDNA (Promega) aliquots after various heat fragmentation times shown adjacent to ImageJ gel analysis of same lanes, molecular weight indicated in bp. Mode fragment size of each smear, as measured with ImageJ, indicated in blue. (B,D,F,H) Agilent 180 K array results of self-hybridizations using reference gDNA (left) and characterized by matching fragment size distributions (B;250/250, D;315/315, F;400/400, H;525/525). Log2 ratios for signal intensities of differentially labeled aliquots (cy5/cy3) are plotted for probes corresponding to chromosome 1 (green;log2ratio<−0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). Data quality was assessed by dLRsd. (I) Mean dLRsd of duplicate (n = 5) or triplicate (n = 2) size-matched self-hybridizations representing seven fragment size distributions plotted by mode fragment length (225, 250, 315, 400, 525, 625, and 680 bp). Error bars indicate SEM.

Mentions: Prior studies have indicated that experimental samples with fragment size distributions less than 300 bp may be a source of inconsistent aCGH performance [9], [16], [19], [24]. Given that matching fragment and reference DNA sizes improves results and might alter baseline performance, we sought to re-evaluate what the optimal fragment size might be under size-matched conditions. To test this we performed additional self-hybridizations using the reference gDNA sample and generated a spectrum of size distributions by varying heat fragmentation times. In total, 16 size-matched self-hybridizations representing seven unique size distributions (range ≈ 200–700 bp; mode fragment lengths ≈ 225, 250, 315, 400, 525, 625, and 680 bp), were measured in duplicate (n = 5) or triplicate (n = 2). In contrast to the size mismatched pairs shown in Figure 1C, D, E, F, G, H, all self-hybridizations between samples with matched fragment sizes yielded data within the acceptable range (dLRsd <0.3), regardless of length of the DNA fragments (Figure 2). We did however observe a significant correlation between decreased dLRsd and increased mode fragment size (r = −0.85, p = 0.015) (Figure 2) with optimal data quality achieved at mode fragment sizes greater than 400 bp (Figure 2I). Overall we observed that optimal aCGH data quality is produced with DNA fragment distributions of paired samples of similar sizes and mode fragment size greater than or equal to 400 bp.


DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues.

Craig JM, Vena N, Ramkissoon S, Idbaih A, Fouse SD, Ozek M, Sav A, Hill DA, Margraf LR, Eberhart CG, Kieran MW, Norden AD, Wen PY, Loda M, Santagata S, Ligon KL, Ligon AH - PLoS ONE (2012)

Determination of optimal size among matched DNA fragment size distributions.(A,C,E,G) Agarose gel electrophoresis of reference gDNA (Promega) aliquots after various heat fragmentation times shown adjacent to ImageJ gel analysis of same lanes, molecular weight indicated in bp. Mode fragment size of each smear, as measured with ImageJ, indicated in blue. (B,D,F,H) Agilent 180 K array results of self-hybridizations using reference gDNA (left) and characterized by matching fragment size distributions (B;250/250, D;315/315, F;400/400, H;525/525). Log2 ratios for signal intensities of differentially labeled aliquots (cy5/cy3) are plotted for probes corresponding to chromosome 1 (green;log2ratio<−0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). Data quality was assessed by dLRsd. (I) Mean dLRsd of duplicate (n = 5) or triplicate (n = 2) size-matched self-hybridizations representing seven fragment size distributions plotted by mode fragment length (225, 250, 315, 400, 525, 625, and 680 bp). Error bars indicate SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376148&req=5

pone-0038881-g002: Determination of optimal size among matched DNA fragment size distributions.(A,C,E,G) Agarose gel electrophoresis of reference gDNA (Promega) aliquots after various heat fragmentation times shown adjacent to ImageJ gel analysis of same lanes, molecular weight indicated in bp. Mode fragment size of each smear, as measured with ImageJ, indicated in blue. (B,D,F,H) Agilent 180 K array results of self-hybridizations using reference gDNA (left) and characterized by matching fragment size distributions (B;250/250, D;315/315, F;400/400, H;525/525). Log2 ratios for signal intensities of differentially labeled aliquots (cy5/cy3) are plotted for probes corresponding to chromosome 1 (green;log2ratio<−0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). Data quality was assessed by dLRsd. (I) Mean dLRsd of duplicate (n = 5) or triplicate (n = 2) size-matched self-hybridizations representing seven fragment size distributions plotted by mode fragment length (225, 250, 315, 400, 525, 625, and 680 bp). Error bars indicate SEM.
Mentions: Prior studies have indicated that experimental samples with fragment size distributions less than 300 bp may be a source of inconsistent aCGH performance [9], [16], [19], [24]. Given that matching fragment and reference DNA sizes improves results and might alter baseline performance, we sought to re-evaluate what the optimal fragment size might be under size-matched conditions. To test this we performed additional self-hybridizations using the reference gDNA sample and generated a spectrum of size distributions by varying heat fragmentation times. In total, 16 size-matched self-hybridizations representing seven unique size distributions (range ≈ 200–700 bp; mode fragment lengths ≈ 225, 250, 315, 400, 525, 625, and 680 bp), were measured in duplicate (n = 5) or triplicate (n = 2). In contrast to the size mismatched pairs shown in Figure 1C, D, E, F, G, H, all self-hybridizations between samples with matched fragment sizes yielded data within the acceptable range (dLRsd <0.3), regardless of length of the DNA fragments (Figure 2). We did however observe a significant correlation between decreased dLRsd and increased mode fragment size (r = −0.85, p = 0.015) (Figure 2) with optimal data quality achieved at mode fragment sizes greater than 400 bp (Figure 2I). Overall we observed that optimal aCGH data quality is produced with DNA fragment distributions of paired samples of similar sizes and mode fragment size greater than or equal to 400 bp.

Bottom Line: While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations.Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues.Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

Show MeSH
Related in: MedlinePlus