Reference alignment of SNP microarray signals for copy number analysis of tumors

Abstract

A new procedure to align single nucleotide polymorphism (SNP) microarray signals for copy number analysis is proposed. For each individual array, this reference alignment procedure (RAP) uses a set of selected markers as internal references to direct the signal alignment. RAP aligns the signals so that each array has a similar signal distribution among its reference markers. An accompanying reference selection algorithm (RSA) uses genotype calls and initial signal intensities to choose two-copy markers as the internal references for each array. After RSA and RAP are applied, each array has a similar distribution of signals of two-copy markers so that across-array signal comparisons are biologically meaningful. An upper bound for a statistical metric of signal misalignment is derived and provides a theoretical basis to choose RSA-RAP over other alignment procedures for copy number analysis of cancers. In our study of acute lymphoblastic leukemia, RSA-RAP gives copy number analysis results that show substantially better concordance with cytogenetics than do two other alignment procedures.

Availability: Documented R code is freely available from www.stjuderesearch.org/depts/biostats/refnorm.

Fig. 1.

Alignment and segmentation results for a hyperdiploid tumor. (A) The quantiles of QA signals for markers on the amplified chromosomes (black dashed line) and chromosomes with no cytogenetic abnormality (solid black line) of a hyperdiploid tumor's array against the corresponding quantiles of QA signals for all markers of a female control tissue array. A diagonal gray line along y=x is included for reference. (C) The results of segmenting the standardized differences (Supplementary Materials, Section A) computed from QA signals. The x-axis represents markers (ordered by chromosome and position), and the y-axis represents the standardized differences. Each gray point represents the standardized difference for one marker, and the thick horizontal lines represent the median of the standardized differences in the determined segments. (B, D) panels show analogous results for the same tumor using RSA-RAP signals. RSA-RAP signals and QA signals are on different scales (A, B) because RSA-RAP maps signals to the targeted normal (0,1) distribution and QA maps to the empirically defined distribution of Equation (1).

Fig. 2.

ROC-type curves. (A–D) the concordance of each alignment procedure's results with cytogenetics across all tumor arrays as a function of the threshold γ among markers on amplified chromosomes, deleted chromosomes, chromosomes with no cytogenetically detected abnormality and all markers on chromosomes in one of these three categories. The solid black, dashed black, solid gray and dashed gray lines give the results for RSA-RAP, cyto-RAP, QA and ISA, respectively.