Normalization of array-CGH data: influence of copy number imbalances
© Staaf et al; licensee BioMed Central Ltd. 2007
Received: 15 June 2007
Accepted: 22 October 2007
Published: 22 October 2007
High-resolution microarray-based comparative genomic hybridization (CGH) techniques have successfully been applied to study copy number imbalances in a number of settings such as the analysis of cancer genomes. For normalization of array-CGH data, methods initially developed for gene expression microarray analysis have, in general, been directly adopted and used. However, these methods are designed to work under assumptions that may not be valid for array-CGH data when copy number imbalances are present. We therefore sought to investigate the effect on normalization imposed by copy number imbalances.
Here we demonstrate that copy number imbalances correlate with intensity in array-CGH data thereby causing problems for conventional normalization methods. We propose a strategy to circumvent these problems by taking copy number imbalances into account during normalization, and we test the proposed strategy using several data sets from the analysis of cancer genomes. In addition, we show how the strategy can be applied to conveniently define adaptive sample-specific boundaries between balanced copy number, losses, and gains to facilitate management of variation in tissue heterogeneity when calling copy number changes.
We highlight the importance of considering copy number imbalances during normalization of array-CGH data, and show how failure to do so can deleteriously affect data and hamper interpretation.
Microarray-based techniques for genome-wide investigation of copy number aberrations (CNAs) have recently gained much attention. Initially employing arrays developed for gene expression analysis , or low-density arrays produced from large-insert genomic clones such as bacterial artificial chromosomes (BACs) , the application has evolved rapidly. Currently, specialized high-density arrays with oligonucleotide probes or probes derived from BAC clones are predominately used. Two-channel array-based comparative genomic hybridization (aCGH) is a direct successor to conventional metaphase CGH . In both cases, DNA from two samples are differentially labeled with fluorescent dyes and co-hybridized to immobilized genomic capture probes. By use of aCGH, DNA derived from tumor tissue can be compared with reference DNA, e.g., normal whole blood DNA, and genomic imbalances can effectively be investigated. The main advantage of aCGH over conventional CGH is the increased resolution achieved by microarrays with a large number of individual probes, routinely up to hundreds of thousands, covering the entire genome . The power of aCGH has been demonstrated in tumor studies [5–8], as well as in the field of clinical genetics , and the basis of the technique is reviewed elsewhere . In essence, relative ratios of copy number between two DNA samples are obtained by comparing the two fluorescent signal intensities for each probe under the assumption that intensities reflect the amount of corresponding genomic DNA in the respective sample.
Using self-self comparisons, in which a sample is compared with itself, it has been observed that other forms of technical bias, e.g., spatial- or plate bias, exist that can skew measured M values enough to revoke the validity of the aforementioned normalization methods . Both methods have therefore been implemented in ways that include stratification of M values in groups of data that are individually subjected to the correction. Stratification can be performed based on, e.g., spatial probe location, or probe source . The general thought is that stratification will result in groups, i.e., populations, of data in which the validity of the normalization method is upheld. It has also been observed that the assumptions, required for conventional normalization methods to work, can fail as a result of a true biological distribution of M, e.g., in situations where the majority of probes measure true differences between compared samples .
We here highlight a well known and commonly displayed property of tumor cells, namely the presence of biologically true CNAs. Figure 1b shows a genome plot of raw M values obtained by aCGH of a female breast cancer tumor xenograft  compared with male normal whole blood DNA. In the genome plot, M is plotted as a function of the genomic location of the probe sequence. In figure 1b, several genomic regions with different and discrete M can readily be observed. We sought to investigate the effect on normalization imposed by this property of aCGH data. We show that this property results in consequential drawbacks when using conventional normalization methods and propose a strategy that incorporates any populations present in the data into the normalization.
The proposed strategy can be integrated with any of several existing normalization methods and results in improved data quality. Also, spatial effects resulting in non-biological, but relevant, populations that can bias normalization are handled when calculating corrections. We also note that part of the procedure can be applied to assign adaptive sample-specific thresholds for calling copy number changes. The proposed normalization strategy, as well as the adaptive sample-specific level scaling, provides powerful and convenient means for improved copy number analysis using aCGH.
Results and Discussion
This study is outlined as follows with results and discussion presented accordingly. To investigate the influence of copy number imbalances on normalization we first created a set of mimicked data representing states of an increasing fraction of genomic gain. Using the mimicked data we demonstrate the effects of gain on normalization using Median and Lowess. We then evaluated an alternative normalization strategy in which data is stratified into separate populations representing gain and balanced copy number respectively. Whereas mimicked data provide prior knowledge facilitating stratification, most experiments lack this information. Therefore, we developed a method for stratification of data and evaluated the method using previously characterized cases. By applying our procedure for stratification and normalization to tumor specimens on different aCGH platforms we compare performance with standard methods. We investigate the implication of technical spatial effects and propose a strategy for improved normalization. In addition, we evaluate the possibility to apply our method to assess noise levels in data and assign sample-specific thresholds for detection of copy number imbalances.
Normalization of aCGH data using Median
Genomic imbalances correlate with intensity in aCGH data
Normalization of aCGH data using population-based intensity-based lowess
We sought to develop a method that corrects for intensity dependence of M due to technical bias while retaining intensity dependence of biological relevance. We reasoned that if we could stratify aCGH ratios from an experiment with respect to copy number populations, we could use this information to circumvent the drawbacks with Lowess. One way to do this would be to run Lowess on one selected population and then apply the resulting correction line on all M values. We refer to this general strategy of considering copy number populations when using Lowess as population-based intensity-based lowess (popLowess). Applying popLowess would serve two purposes. Firstly, data would be centered at a copy number population rather than a mean or median of a mixture of different and possibly diverse copy number levels. Secondly, correlations between M and A related to technical bias would be identified and corrected for without affecting the intensity dependence due to different copy numbers. To test this strategy, we subjected the mimicked XXX/XX data sets to popLowess. Since we had prior knowledge about this case we could stratify values into copy number populations based on chromosome mapping. All values for autosomes were considered to comprise one population and all values from the X chromosome another.
Stratification of M values into copy number populations
Comparison of popLowess enriched population assignment to karyotyping data for eight hyperdiploid cases 
Gain (fraction of karyotype called)***
A procedure for normalization of aCGH data using popLowess
Once data is stratified into sets of enriched copy number populations we can select one, e.g., the largest, to perform Lowess normalization on. The generated correction curve must be generalized to cover the full range of A allowing for correction of all M values (Figure 6e). This procedure will ensure that the lowess derived correction line trails one population and remains unaffected by adjacent ones. We refer to this action as popLowess-o (where the letter o is a mnemonic for one) as it makes use of one population to derive a correction line for all data. The complete procedure of data stratification and popLowess normalization is shown in figure 5, steps 1–8. Naturally, once data is stratified alternative variants of calculating normalization corrections are imaginable. For example, one could fit lowess lines to each population and correct them individually or one could individually center populations and then use the combined data to create a lowess derived correction line. We refer to these alternatives as popLowess-i (where the letter i is a mnemonic for individual) and popLowess-c (where the letter c is a mnemonic for common) respectively. The latter alternative has the added advantage of reducing the degree to which the correction line needs to be extrapolated to cover the full range of A. Both alternatives require an additional step to center a selected copy number population at M = 0. The variants popLowess-o and popLowess-c rely on that the intensity-based curvature in M-A space is reasonably shared between populations.
Selecting a population to represent intrinsic copy number
The normalization procedure presented herein will center a population with unknown copy number at M = 0. The rationale for selecting an appropriate population for this purpose can differ depending on samples analyzed and the aim of a project. For instance, in the field of cytogenetics, gains and losses in tumors are by convention described as net changes relative to intrinsic balanced copy number, i.e., relative ploididy. As the number of centromeres determines ploidity, a parallel rationale would be to relate imbalances relative to the largest identified population and therefore center this population at M = 0. However, in some applications it might be more appropriate to relate imbalances to a normal diploid state. Thus, selecting a population to center data at can include using prior knowledge about regions with known copy number or selecting the middle population out of three, if present. Irrespectively of preferences of how data best be centered, the proposed popLowess procedure will alleviate the normalization problems related to mixed copy number populations. Importantly, when performing focused aCGH with specialized arrays that do not cover the entire genome, or comprise probes with a disproportioned focus on specific genomic regions, even CNAs that affect a minor part of the genome can introduce a significant correlation between copy number and intensity, and can result in misinterpretations of how a given ratio level relate to copy number.
Application to tumor specimens on different aCGH platforms
Comparison of popLowess strategy to standard normalization methods
We set out to test if the popLowess strategy could systematically reduce variation in M within copy number populations in different aCGH data sets. We hypothesized that when correction curves cross, or not accurately track, copy number populations; or when intensity-based curvature is not properly addressed, a larger variation in M is obtained after normalization. To this aim, we compared the performance of the popLowess strategy versus Median and Lowess using seven different aCGH data sets (data sets 1–6, 8). The data sets cover three different types of aCGH platforms hybridized with a variety of cell line and tumor samples displaying a large variation of CNAs.
We used the strategy in figure 5 to identify copy number populations in each of the data sets. We then normalized each data set in parallel using popLowess, Lowess, and Median. After normalization, we calculated standard deviations of M for each identified population for each method and compared results.
Comparison of effect on population variance between different normalization strategies
P-values for data sets
Nbr of samples
BAC 32 K
BAC 32 K
BAC 32 K
BAC 1 Mb
Agilent 244 K
Agilent 44 K
BAC 32 K
popLowess vs Lowess
popLowess vs Median
Since we do not have prior knowledge of CNAs in most of the cases we cannot evaluate variation within confirmed genomic regions of similar copy number. Therefore, one could argue that the better performance of popLowess, resulting in lower variation within populations when compared with conventional normalization, is biased by the fact that populations are inferred from the data. However, from looking at the data in table 1, and at the genome plots in figure 7 (panel g and h) we note that the identified populations reflect regions with discrete copy number levels. Therefore, we argue that decreased intra population variation is beneficial to both interpretation and downstream analysis and provides improved data quality.
Presence of technical artifacts in array data resulting in correlation between M and spatial probe location on the array is a well-known and previously described phenomenon. We focused on two plausible consequences of such spatial effects in aCGH data. Firstly, affected values can introduce populations that compromise normalization in the same way as copy number populations. Secondly, affected values will be incorrectly scaled compared to non-affected.
We reasoned that ratios biased by spatial artifacts are controlled for by our proposed popLowess strategy as it filters outlier data guided by genomic mapping. Thus, when calculating an intensity dependent correction for normalization, our strategy would not be compromised by spatial bias as affected values are disregarded together with values from break points, high-level amplifications, and homozygous deletions. On the other hand, popLowess does not correct for spatial effects and affected values would remain incorrectly scaled after normalization even if the intensity bias is removed.
As the proposed popLowess strategy does not correct for spatial effects, we reasoned that a pre-normalization step might be appropriate for data displaying spatially related bias in order to properly scale affected values. This could be accomplished by applying one of many available spatial correction methods [15–17], or variations thereof, prior to popLowess. However, since we have shown that genomic imbalances correlate with intensity, we are cautious about addressing spatial effects using pre-normalization algorithms that are intensity-based.
To test our reasoning we applied popLowess to data set 7. Samples in this set have little to no genomic alterations but the data display variation in M-A curvature and spatial effects. Data set 7 was normalized using popLowess, block-based Median followed by popLowess, or block-based Lowess followed by popLowess. For popLowess, by itself or in combination with a pre-normalization step, a merge cluster criteria of 0.3 in M was employed to account for the presence of only two copy number populations.
Effect of pre-normalization to correct spatial bias prior to applying popLowess
pre-normalization by block-based Median***
pre-normalization by block-based Lowess****
XY vs XY
XY vs XY
XX vs XX
XX vs XX
XXX vs XX
XX vs XY
XXXX vs XX
XXX vs XY
XXXX vs XY
We conclude that the proposed popLowess strategy is robust in the sense that it can handle the presence of otherwise deleterious populations without relying on them. We also conclude that, whereas popLowess is inert to spatial effects, in the sense that it does not compromise calculation of an intensity dependent correction, a pre-normalization step that correct for spatial bias is warranted.
Adaptive sample-specific thresholds for calling copy number change
During development of the popLowess strategy, we recognized that the sample-specific cut-off value (Figure 5, step 3) could be used to assess noise level in data and to assign thresholds for copy number imbalances on a sample-specific basis. Several reports [5, 8, 22, 23] have utilized global thresholds in M for calling CNA as gains or losses. These thresholds are assigned by adding/subtracting a value in M from a base line typically at M = 0. Determining suitable thresholds may be problematic in large sample sets with samples of varying quality and heterogeneity, often the case for tumor studies , and may result in setting too conservative thresholds for certain samples in order to avoid erroneous CNA calls. Deriving sample-specific threshold values scalable for desired stringency in an automated fashion is then of relevance.
A parallel can be made to the derivative log ratio spread (DLR) value calculated by the Agilent CGH Analytics software. The DLR-value can be used to assess hybridization quality and provide a sample scalable threshold for calling CNAs using, e.g., the Z-scoring algorithm in the CGH Analytics software.
Normalization affects downstream analysis
We show that the presence of copy number populations in aCGH data deleteriously affects normalization using curve-generating algorithms such as intensity-based lowess and may cause erroneous centering of data. We demonstrate that genomic imbalances correlate with intensity in aCGH data and therefore must be accounted for during normalization in order to correct for intensity dependence of M due to technical bias while retaining intensity dependence of biological relevance. Here we propose a population-based normalization strategy that accounts for the presence of copy number populations. We show that benefits of a population-based normalization approach are clearly recognized for data displaying numerous CNAs. We also demonstrate that the proposed procedure can be applied to assign adaptive sample-specific thresholds for calling copy number changes. We appreciate that the suggested strategy represents only one conceivable way of implementing population-based normalization and that any implementation that effectively discerns copy number populations in aCGH data, whether utilizing prior knowledge regarding samples or inference from the data itself, could be used. In addition, once copy number populations are identified, this information can be used in a variety of ways to circumvent highlighted problems related to conventional normalization of aCGH data. Taken together, we demonstrate that copy number populations in aCGH data should be accounted for during normalization and that the proposed normalization strategy, as well as the adaptive sample-specific level scaling, provides powerful and convenient means for improved copy number analysis using aCGH.
We used eight data sets derived from BAC arrays and from Agilent 244 K oligonucleotide CGH arrays to evaluate normalization methods. Data set 1 consists of seven breast cancer cell lines analyzed using tiling 32 K BAC arrays . Data set 2 consists of 28 lung cancer cell lines analyzed using tiling 32 K BAC arrays . Data set 3 consists of ten breast cancer cell lines analyzed using tiling 32 K BAC arrays . Data set 4 consists of 52 breast cancer tumors analyzed in dye-swaps on 1 Mb BAC arrays . Data set 5 consists of 8 breast cancer tumors and one dye-swap analyzed using Agilent 244 K oligonucleotide CGH arrays . These tumors displayed DLR values between 0.196 and 0.364 when analyzed with Agilent CGHAnalytics software ver 3.4.27 . Data set 6 was created from data set 5 by matching the oligonucleotide probe IDs from the 244 K arrays to the Agilent 44B probe IDs available through Agilent eArray , thus creating a virtual 44 K oligonucleotide CGH array. Of 42,447 genome-mapped probe IDs on the 44B array, 41,599 were found on the 244 K arrays (98%). Data set 7 consists of nine hybridizations of chromosome X aberrant cell lines with karyotype 47, XXX and 48, XXXX, and male 46, XY and female 46, XX samples in various combinations . Samples in data set 7 are expected to display a normal karyotype for chromosomes 1–22. Data set 8 consists of eight hyperdiploid childhood ALL cases analyzed using tiling 32 K BAC arrays .
Pre-filtering and conventional normalization of aCGH data
All data sets were loaded into BioArray Software Environment (BASE)  for analysis. Positive and non-saturated spots were background corrected using the median foreground minus the median background signal intensity for each channel and log ratios (M) were calculated from the background corrected intensities. In all analysis we used M = log2(int1/int2) and A = log10(sqrt(int1*int2)), where int1 and int2 are background corrected intensities from the investigated sample and reference, respectively. Data sets 1–4 and 7–8 were filtered for signal-to-noise ratio for each spot in both channels according to published reports and the remaining data sets for signal-to-noise ratio > 5 in both channels before BASE implemented software plug-ins of the different normalization strategies were employed. A lowess smooth factor of 0.33, delta of 0.1, and four iterations were used for standard Lowess, popLowess and block-based lowess normalization. Block group size was set to 1 for all block-based normalizations.
Population-based intensity-based lowess
A schematic overview of the proposed popLowess normalization strategy is shown in figure 5. The approach is applied on a per sample basis starting with genomic mapping and raw intensities (int1 and int2) for N probe IDs (step 1, Figure 5). The probes are sorted according to genomic position and M and A are calculated for each probe (step 2, Figure 5). Next, a standard deviation in M is calculated for each probe in sliding windows of user-defined size along the genome. The resulting distribution of N standard deviations is subjected to a cut-off criterion generating K probes with standard deviations < cut-off for continued population analysis (step 3, Figure 5). A moving window size of 11 probes was used and the median of the standard deviation distribution was used as cut-off value. This selection criterion is sample adaptive avoiding problems with using a global cut-off criterion. The K selected probes are next segmented on a per chromosome basis using, e.g., the CGHplotter algorithm  or the faster circular binary segmentation (CBS) algorithm  (step 4, Figure 5). Herein, the segmentation algorithm proposed by Autio et al. was used with the constant for computing the number of changes (c-parameter) set to 10 . Segmented values are used to cluster the K probes into three distinct clusters by means of robust k-means clustering (step 5, Figure 5). After clustering, there is an option to merge clusters with cluster centers close to each other. Merging is typically useful for samples not displaying three populations, e.g., samples with 1 or 2 copy number populations. When indicated, a merge cluster criterion of 0.2 or 0.3 in M was used. The resulting data consists of 1–3 distinct populations of data that contains information about the genomic mapping, M, and A for each probe. The largest population is selected for lowess normalization  generating a population specific correction curve (step 6, Figure 5). The correction curve is next extrapolated to the entire range of A and used to correct M for all N reporters similar to Lowess (step 7, Figure 5). The extrapolation is done conservatively in the end points of A by using the first/last data point of the population specific correction curve to level out the global correction curve horizontally in the M-A plot thereby moderating the impact of extreme points or missing values. After lowess correction, one population is selected as the center population and all data is shifted such that this population obtains median M equal to 0. Selection of a center population can be based on different assumptions. Finally, the normalized int1 and int2 intensities are returned (step 8, Figure 5). By not segmenting the entire set of observations, and by setting the crucial segmentation parameters for detecting breakpoints in the lower scale, speed is gained while still retaining robustness as long as the standard deviation cut off is not set too low. The purpose of segmentation is to refine large regions with identical copy number and not to detect small complex copy number alterations.
Comparison of normalization methods
For comparisons, the R implemented lowess function was used to create lowess-normalized data. For each identified population (step 1–5, Figure 5) in every sample in data sets 1–6 and 8, the standard deviations in M of the reporters in the population after Lowess, popLowess, and no normalization (equal to Median) were calculated separately. The number of populations in a data set for which the popLowess strategy rendered a lower standard deviation compared to the competitor was calculated. To evaluate if popLowess resulted in a significant number of populations with lower standard deviations, one sided p-values were calculated using the binomial distribution with p = 0.5. This binomial test corresponds to the null hypothesis that lower standard deviations for popLowess are obtained by chance. This comparison was done both when studying all populations as a whole and for each population individually.
Sample adaptive gain/loss thresholds
Sample adaptive thresholds for calling gain or loss can be generated by performing steps 1–3 in Figure 5 using the same form of data input and standard deviation cut-off criteria. The identified standard deviation cut-off value can be scaled by multiplicative factors to generate sample specific gain/loss thresholds of desired stringency for downstream applications, e.g., calling CNAs after segmentation. Before creating sample adaptive thresholds, data was pre-filtered and normalized using the popLowess strategy. Sample adaptive thresholds for the Ca13928 breast tumor were created before and after a smoothing window of 250 kBp size for 32 K BAC data and 50 kBp for Agilent 244 K data. Thresholds were estimated using a chromosomal moving window of size 1% of the total probe number for each chromosome separately and the standard deviation cut-off value was selected as the median of the standard deviation distribution. The cut-off value was scaled by a factor 2 to create the ± thresholds in M displayed in figure 9.
Availability and requirements
Project name: popLowess
Project home page: http://baseplugins.thep.lu.se/wiki/se.lu.onk.popLowess
Operating system(s): Platform independent
Programming language: R
License: GNU GPL
List of abbreviations
acute lymphoblastic leukemia
bacterial artificial chromosome
BioArray Software Environment
comparative genomic hybridization
copy number aberration
copy number variation
Fluorescence in situ hybridization
Inter Quartile Range
Global intensity-based lowess normalization
Global median normalization
population-based intensity-based lowess normalization
Spectral karyotyping technique
We wish to thank Patrik Edén and Mattias Höglund for helpful comments on the manuscript. This work was supported by the Knut and Alice Wallenberg Foundation via the SWEGENE program (JS and JVC), the Swedish Cancer Society (GJ), the American Cancer Society (GJ and JVC), John och Augusta Perssons stiftelse (GJ and JVC), and the Swedish Foundation for Strategic Research through CREATE Health – the Lund Strategic Centre for Clinical Cancer Research (MR).
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