Biomedical Engineering Reference
In-Depth Information
copy number analysis, in particular for the GoldenGate assay with fewer SNPs (6000 SNPs)
compared with 100 000 or 317 000 SNP Infinium arrays.
aa
This software can identify the individual beads and measure the intensity of each bead. In
general, it is useful to organize the files created by BeadScan by putting them into separate
subdirectories for each experiment.
bb
These settings also create the following files: tiff: scanned image of sample; csv: summary
values per probe - intensity, standard deviation, number of beads; txt: values for each bead -
intensity, position on tif.
cc
Illumina provides two software packages that can perform genotyping on the scanned images.
Gencall was the original application for genotyping and was followed by BeadStudio, which is an
integrated package for performing all kinds of data analysis for Illumina arrays. The algorithm
for genotyping differs between the packages. We prefer to use the results of GenCall as the
input for copy number analysis. If genotyping is performed with the Illumina Gencall software
then the csv files are needed to obtain copy number values.
dd
Beadstudio produces a report file with a specifiable number of result fields. For the copy
number analysis the following fields are needed: GC Score Allele1 - AB Allele2 - AB GT Score X
Raw Y Raw. The report file will contain the chosen values for each sample and probe.
ee
This calculation consists of three stages: normalizeBetweenAlleles.SNP() Performs quantile
normalization between both colors of a sample. This is allowed because the frequencies of both
alleles throughout a sample are nearly identical in practice. This action also neutralizes any
dye bias. normalizeWithinArrays.SNP() scales each sample using the median of the high-quality
heterozygous SNPs as the normalization factor. Genomic regions that show copy number
alterations are likely to show LOH, or are harder to genotype leading to a decrease quality score
of the call. normalizeLoci.SNP() scales each probe using the normal samples in the experiment,
assuming that these samples are diploid, and have a copy number of two.
ff
This can be the file produced by the Illumina genotyping software, but it is useful to
include more information on the individual samples here; for example, experimental groups,
normal/tumor tissue. The format for these data is explained in detail in the help pages for
the package. The data quality should be checked. The overview of average intensities for both
channels as laid out on the physical device has proved helpful to detect technical anomalies.
For the GoldenGate assay both channels should have an average intensity above 1250.
gg
Various plots of raw or smoothed copy number data can be obtained using the Quantile
smoothing software, such as: smoothed intensities from all chromosomes for a number of
samples, all samples in an experiment indicating regions of gain, loss and LOH, for individual
chromosomes, or a BAC-array-like plot for each sample in an experiment.
hh
A gain is called where the 25th percentile line exceeds the 2N line and a loss is called where
the 75th percentile is below the diploid line.
1.2.3 Multiple ligation-dependent probe amplification (MLPA)
The MLPA technique was first described by Schouten
et al
. [7] in 2002 and has become a
rapidly growing technique used in the detection of aberrations in genes related to various
diseases. MLPA is a multiplex technique for determining copy numbers of genomic DNA
sequences [32] and promoter methylation status [33], as well as mRNA profiling [34]. While
genomic profiling is becoming increasingly important in both the research and diagnostic
setting, routine detection methods for exon deletions and duplications are still lacking.
