A Technical Guide to Aneuploidy
Calling with 24sure Single Channel
Introduction
X-Separation
Biopsy Types
Potential Causes of Noise
Process for Calling
Examples of Calling Challenging Profiles
Limitations of Standard Algorithms and the Green Line
Overview of Calling 24sure Single Channel Assays
Calling Individual Changes
Summary of Expected X/Y Chromosome Scenarios
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15056973 Rev. A
August 2014
1
2
4
10
12
13
15
16
17
18
19
This document and its contents are proprietary to Illumina, Inc. and their affiliates (collectively, "Illumina"), and are intended
solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other
purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise
communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does
not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by
this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND
DAMAGE TO OTHER PROPERTY.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE
THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION
WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S).
FOR RESEARCH USE ONLY© 2014 Illumina, Inc. All rights reserved.
Illumina, IlluminaDx, 24sure, BaseSpace, BeadArray, BeadXpress, BlueFish, BlueFuse, cBot, CSPro, CytoChip, DASL,
DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X,
Infinium, iScan, iSelect, MiSeq, MiSeqDx, NeoPrep, Nextera, NextSeq, NuPCR, SeqMonitor, Solexa, TruGenome,
TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, VeriSeq, the pumpkin orange color, and the Genetic Energy
streaming bases design are trademarks of Illumina, Inc. in the U.S. and/or other countries. All other names, logos, and other
trademarks are the property of their respective owners. SurePlex contains technology developed and manufactured by Rubicon
Genomics Inc., Ann Arbor, Michigan, USA. Protected by US Patent 8,206,913; and pending.
2
Part # 15056973 Rev. A
24sure is a proven and reliable technology for 24 chromosome copy number analysis in
single or a few cells including polar bodies, blastomeres, and trophectoderm biopsies.
The application is known as preimplantation genetic screening (PGS) for aneuploidy. In
most cases, data interpretation and calling is straightforward. However, as with other
genomic screening technologies, the quality of DNA and precise experimental conditions
can affect the quality of results. It is important to understand these potential effects.
When calling more challenging samples, it is also important to interpret the data with
reference to the type of biopsy and the biologically possible genetic scenarios (chromatid
or chromosome imbalances, mosaicism).
While the BlueFuse Multi software provides automated calling functionality, these calls
are not definitive, particularly for more complex samples. Laboratories must understand
the calling process in detail so that they can consistently obtain the most reliable results.
This document provides guidelines for calling, with particular emphasis on more
difficult cases. It does not provide trouble-shooting or protocol advice. The focus is on
making the best calls from the available data.
Terminology
This field presents some terminology challenges. Euploid is often used to mean a normal
chromosome complement of 23 pairs. Technically, euploid indicates an exact multiple of
the haploid chromosome complement (23 individual chromosomes or n), and therefore
46 chromosomes (23 pairs or 2n). However, euploid can equally indicate 23
chromosomes (n) or 69 chromosomes (3n).
Array technologies, such as 24sure, use techniques that compare quantities of DNA from
a sample and a reference and the data are plotted as a ratio on the log scale around zero.
When a sample contains the correct complement of chromosomes, the data are plotted
around zero on the log2 scale. However, this data representation could indicate euploid
results of n, 2n, or 3n, but not necessarily a normal or diploid genome.
Terminology is further complicated when discussing polar bodies. Polar body 1 normally
contains a complete set of individual chromosomes (n), each comprising of a
chromosome with two sister chromatids. Polar body 2 also normally contains a complete
set of individual chromosomes (n). However, each chromosome in polar body 2 contains
a single chromatid.
In general, when discussing the number of copies of an individual chromosome, the
terminology uses the "somy" ending: monosomy (one copy), disomy (two copies), trisomy
(three copies), tetrasomy (four copies), polysomy (many copies). When referring to copies
of a complete set or complement of chromosomes, the ending used is "ploid": diploid
(2n), triploid (3n), tetraploid (4n), polyploid (multiples of n greater than 3n), euploid
(multiple of n), aneuploid (any state that is not euploid).
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
1
Introduction
Introduction
X-Separation
For technical reasons, there is a difference between the theoretical ideal maximum
chromosome copy number change and the numerical log2 ratio actually observed in a
24sure assay. For example, a trisomy 21 in a blastomere (a 3:2 biological ratio) might
have a log2 ratio of 0.4 in a 24sure assay, rather than the theoretical 0.58 (log2(3/2)). The
precise numbers can vary with protocol conditions, so it is important to calibrate an
assay against an internal control of known copy number.
One advantage of 24sure processed using the single channel protocol is that each sample
is compared with both a male and a female reference. Therefore, the X chromosome can
be used as a natural internal control for calibration. Specifically, when a sample with a
normal chromosome X complement is compared with a reference of the opposite sex (a
sex-mismatch), two scenarios are possible, a 1:2 or 2:1 biological ratio. These scenarios
occur because a male has one X chromosome and a female has two. The observed
magnitude of the X chromosome log2 ratio, in either the + or – direction on the log2 ratio
scale, is called the X-separation in this document. It provides a direct mapping between
a theoretical biological ratio of 2:1 (female versus male, log2(2/1) = 1) or 1:2 (male versus
female, log2(1/2) = -1) and the actual observed ratio in 24sure.
BlueFuse Multi attempts to match the sex of the sample. The sex matched data are
plotted as green dots on the fused chart (combined) display. The sex mis-matched data
points are also displayed. If the predicted mismatch is a female reference, the data points
are displayed as a pink line. If the predicted mismatch reference is male, the data points
are displayed as a blue line. The example in Figure 1 shows a male sample run using a
24sure Single Channel experiment type in BlueFuse Multi. X-separation of this assay is
approximately -0.75, as measured by the pink line showing the sample compared to
female reference. The shift of chromosome 13 is of a similar magnitude, indicating this
data shows a true whole chromosome loss.
Figure 1 Male sample processed as a Single Channel experiment type. Sex matched to male
reference (green line), sex mismatched to a female reference (pink line). Plotted data
indicates a loss of chromosome 13.
X-separation is consistent across samples, provided experimental conditions are
consistent (as is the case within a single batch of 24sure Single Channel assays). This
consistency is important because X-separation can be difficult to resolve definitively in a
sample with an X chromosome abnormality. In this situation, X-separation can be
deduced by considering other samples in the same batch and considering reference
hybridizations.
X-separation does vary with hybridization time, ranging from around 0.4 for a three
hour hybridization to 0.9 for a high-quality overnight hybridization. This variance is not,
by itself, a cause for concern because reliable calls can still be made, provided Xseparation is taken into account.
Protocol errors can also affect X-separation. For example, insufficient Cot-1 DNA or
ineffective washing can drastically reduce X-separation and cause failed experiments
2
Part # 15056973 Rev. A
X-Separation
where aneuploidy cannot be reliably called. An X-separation of, for example, 0.2
indicates a failed experiment.
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
3
Biopsy Types
The objective of copy number calling is to determine which samples have a normal
complement of chromosomes for the cell type being analyzed and which have an
abnormal copy number. Each biopsy type is different and the differences must be
considered when interpreting 24sure data. These differences are summarized in a series
of tables for polar bodies, blastomere, and trophectoderm biopsies, with the format of the
tables explained in Polar Body 1 (PB1).
Polar Bodies
Polar Body 1 (PB1)
A normal complement in PB1 is two for each chromosome – two sister chromatids
should be present.
An abnormal copy number chromosome in PB1 can be zero, one, three, or four sister
chromatids.
Table 1 Polar body 1 (PB1)
Number of
Chromatids
Direction of Change
(Gain/Loss)
Expected Magnitude
of Change
Average log2 ratio
with X-separation of:
0.8
0.6
0.5
0.4
Chromosome
Loss
Chromatid
Loss
0
1
Normal
Copy
Number
2
Loss
Loss
~2x Xseparation
-1.0 and
greater*
-1.0 and
greater*
-1.0
-0.8
Chromatid
Gain
Chromosome
Gain
3
4
-
Gain
Gain
1x Xseparation
0
0.6x Xseparation
1x Xseparation
-0.8
0
0.48
0.8
-0.6
0
0.36
0.6
-0.5
-0.4
0
0
0.30
0.24
0.5
0.4
*In this context, "greater" means that chromosome loss is further from 0, i.e. -1.1, -2.
In Table 1, the average log2 ratio is provided for different levels of X-separation. Refer to
the average log2 ratio for a copy number imbalance of a particular kind.
NOTE
Variability of +/- 20% can occur around these averages, depending primarily on the
quality of the amplification and performance of the assay.
To illustrate the value of Table 1, consider a PB1 experiment with X-separation of 0.6 and
a putative copy number loss on chromosome 3 with a log2 ratio of -0.3. From Table 1, a
chromatid loss is expected to have magnitude similar to X-separation. However, the
putative loss on chromosome 3 is under half the expected ratio for a genuine change and
is therefore an artifact of amplification.
4
Part # 15056973 Rev. A
Biopsy Types
Polar Body 2 (PB2)
A normal copy number in PB2 for each chromosome is one sister chromatid.
An abnormal copy number can be zero, two, three, or four chromatids.
NOTE
Three and four chromatids are seen less frequently in practice.
Table 2 Polar body 2 (PB2)
Chromatid
Loss
Number of Chromatids
Direction of Change (Gain/Loss)
Expected Magnitude of Change
Average log2 ratio with Xseparation of:
0.8
0.6
0.5
0.4
0
Loss
~2x Xseparation
-1.0 and
greater*
-1.0 and
greater*
-1
-0.8
Normal
Copy
Number
1
0
2
Gain
1x Xseparation
2x
Chromatid
Gain
3
Gain
1.58x Xseparation
0
0.8
1.27
0
0.6
0.95
0
0
0.5
0.4
0.79
0.63
Chromatid
Gain
*In this context, "greater" means that chromosome loss is further from 0, i.e. -1.1, -2.
NOTE
For PB2, a chromatid gain results in a higher magnitude change (1x X-separation)
than a chromatid gain in a PB1 (0.6x X-separation).
Polar Body 1 and 2 – Analysis
Abnormalities of copy number in PB1 or PB2 must be considered in combination,
because abnormal copy number in PB1 can balance an abnormal copy number in PB21.
The following theoretical scenarios, where loss/gain of chromatids is equal across a
PB1/PB2 pair from a single oocyte, are all indicative of a normal copy number in the
oocyte.
Table 3 Theoretical PB1 and PB2 scenarios resulting in diploid oocyte
Oocyte
PB1 (# chromatids)
PB2 (# chromatids)
1 (chromatid loss)
2 (chromatid gain)
Normal copy number
3 (chromatid gain)
0 (chromatid loss)
Normal copy number
0 (chromosome loss)
3 (2x chromatid gain)
Normal copy number
2 (normal copy number)
1 (normal copy number)
Normal copy number
Oocytes in the first three scenarios have normal copy number despite aneuploid polar
bodies. It has been shown that a chromosomally normal child can result from an oocyte
with aneuploid polar bodies with reciprocal chromosome imbalances2.
All other scenarios result in an aneuploid oocyte.
Harton GL et al (2011). ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines
for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Hum Reprod.
2011 Jan;26(1):41-6.
1
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
5
Scott RT et al (2012). Delivery of a chromosomally normal child from an oocyte with reciprocal aneuploid polar
bodies. J Assist Reprod Genet. 2012 Jun;29(6):533-7
2
Example 1
This example comprises of three profiles from PB1, PB2, and a blastomere from the same
patient. PB1 is euploid, PB2 has a chromatid gain on chromosome 13, and the
blastomere has a chromosome loss on chromosome 13 as expected.
Figure 2 Profile for PB1, euploid
Figure 3 Profile for PB2, chromatid gain on chromosome 13 (red circle)
Figure 4 Profile for blastomere, chromosome loss on chromosome 13 (red circle)
Example 2
In this example, we show the correction of one chromosome between PB1, PB2, and the
day 3 embryo. PB1 has a chromatid loss on chromosome 15, PB2 has a chromatid gain
on chromosome 15, and the embryo has a normal copy number for chromosome 15 as
expected. The blastomere biopsy in Figure 7 also indicates the loss of chromosome 19
and chromosome 21, as predicted from the PB1 and PB2 profiles.
6
Part # 15056973 Rev. A
Figure 6 Profile for PB2, chromatid gain on chromosome 15 (red circle) and chromatid gain
for 19
Figure 7 Profile for blastomere biopsy, euploid chromosome 15 (red circle) and chromosome
loss for 19 and 21
Blastomeres
A normal copy number blastomere has two copies of each chromosome.
A blastomere with abnormal copy number can have zero (nullisomy), one (monosomy),
three (trisomy), four (tetrasomy), or more copies of one or more chromosomes.
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
7
Biopsy Types
Figure 5 Profile for PB1, chromatid loss on chromosome 15 (red circle) and chromatid gain for
21
Table 4 Blastomere
Number of Chromosomes
Direction of Change (Gain/Loss)
Expected Magnitude of Change
Average log2 ratio with Xseparation of:
0.8
0.6
0.5
0.4
Normal Copy
Number
2
0
Nullisomy
Monosomy
Trisomy
0
Loss
~2x Xseparation
1
Loss
1x Xseparation
-1.0 and
greater*
-1.0 and
greater*
-1.0
-0.8
-0.8
0
0.48
-0.6
0
0.36
-0.5
-0.4
0
0
0.30
0.24
3
Gain
0.6x Xseparation
*In this context, "greater" means that chromosome loss is further from 0, i.e. -1.1, -2.
A day 3 embryo can be mosaic–some blastomeres have a normal chromosome
complement (diploid) while others have an abnormal copy number (aneuploid).
However, mosaicism is not possible in any individual blastomere. Therefore,
chromosomes showing small ratio changes that are inconsistent with the ratio changes
specified in Table 4 are possibly technical artifacts or biological artifacts related to cell
cycle stage, and are not indicative of mosaicism. Figure 8 shows amplification artifacts
on chromosomes 11 and 13, which are not consistent with a genuine imbalance.
Figure 8 Blastomere profile, amplification artifacts on chromosomes 11 and 13 (red circle)
Trophectoderm Biopsy
A trophectoderm biopsy typically has 3–5 cells, but varies from biopsy to biopsy and can
range from 1 to 10 cells or more. Interpretation is the same as for a blastomere. However,
mosaicism is possible because there are multiple cells.
8
Part # 15056973 Rev. A
Biopsy Types
Table 5 Trophectoderm
Number of Chromosomes
Direction of Change (Gain/Loss)
Expected Magnitude of Change
Average log2 ratio with X-separation
of:
No Mosaicism
0.8
0.6
0.5
0.4
Mosaicism of 50%
0.8
0.6
0.5
0.4
Normal
Copy
Number
2
0
Nullisomy
Monosomy
Trisomy
0
Loss
~2x Xseparation
1
Loss
1x Xseparation
-1.0 and
greater*
-1.0 and
greater*
-1.0
-0.8
-0.8
0
0.48
-0.6
0
0.36
-0.5
-0.4
0
0
0.30
0.24
-0.5 and
greater*
-0.5 and
greater*
-0.5
-0.4
-0.4
0
0.24
-0.3
0
0.18
-0.25
-0.2
0
0
0.15
0.12
3
Gain
0.6x Xseparation
*In this context, "greater" means that chromosome loss is further from 0, i.e. -1.1, -2.
Because there are multiple cells in a trophectoderm biopsy and therefore more DNA, the
array profiles for trophectoderm biopsies are less noisy and generally easier to interpret.
However, mosaicism is possible in trophectoderm biopsies. Detection of mosaicism
under 50% is not recommended because of potential DNA amplification effects. The
relevance of mosaicism in predicting embryo viability is also unknown clinically.
Figure 9 follows 24sure screening of a trophectoderm sample. This case is mosaic for
trisomy 2, 8, 10 and 12. Mosaicism is consistent with two out of four cells having
trisomies. You can calculate the legitimacy of the imbalance using Table 5. The Xseparation is approximately 0.6, and the shift on chromosome 2 is approximately 0.25.
By looking at the row where X-separation is 0.6, an imbalance of 0.25 would be
consistent with a trisomy in a mosaic sample.
Figure 9 Screening of a trophectoderm sample, mosaic for trisomy 2, 8, 10 and 12 (red circles)
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
9
Potential Causes of Noise
Most 24sure profiles exhibit low noise and interpretation is straightforward, as shown in
the trisomy 21 blastomere profile in Figure 10.
Figure 10 Trisomy 21 blastomere profile (red circle)
However, noise can be introduced into the assay via a number of technical mechanisms
and it is important to be aware of both causes and effects. The three main causes are
Failed Amplification, Experimental Effects, and DNA Quality.
Failed Amplification
A failed amplification occurs for various reasons, but is most commonly due to lack of
an accessible high-quality cell for amplification. In most cases, a failed amplification can
be detected by observing lack of a smear in an agarose gel run as part of the SurePlex
amplification protocol. However, occasionally, a smear can be present even when
amplification has failed. Amplification failure can be due to non-human contaminating
DNA or occasional self-priming of the SurePlex reaction. These samples have a
characteristic negative control profile with high noise, wavy data, and lack of X/Y
separation as shown in Figure 11. These assays should be failed.
Figure 11 Example of failed amplification profile
Experimental Effects
Protocol errors can affect the quality of 24sure data. However, the most common effects
of protocol errors on results are a reduction in X-separation or an increase in background
noise. Such assays are often still able to be called.
DNA Quality
The “quality” of DNA can affect the clarity of results from 24sure screening and, also
more widely, in a range of non-microarray technologies. DNA quality refers to effects of
10
Part # 15056973 Rev. A
“Poor” quality DNA can lead to increased levels of random noise without bias, wave
patterns in the data, and low magnitude whole chromosome changes. It is important to
be aware of these effects, which can visually appear like chromosome changes at first
glance.
Wave Pattern
In some samples, a slowly varying “wave” is visible. It is commonly seen in
chromosomes 1, 9, 10 and 11 but can also appear elsewhere. This pattern is an
amplification artifact and is biopsy-specific. It is most extreme in single cell assays. An
example of a clearly euploid sample with wave effect is shown in Figure 12.
Figure 12 Euploid profile with wave pattern
Low Magnitude Whole Chromosome Changes (“Step Changes”) from
Blastomere Samples
Whole chromosome step changes can occur as shown in Figure 13. These step changes
appear convincing but can, in most cases, be ruled out due to their low X-separation
magnitude being inconsistent with biological reality.
Figure 13 Blastomere profile with low magnitude whole chromosome changes (red circles)
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
11
Potential Causes of Noise
DNA nicking, incomplete cell lysis, cells undergoing apoptosis, the presence of PCR
inhibitors in media, or protocol deviations that affect lysis or hybridization specificity.
PGS/PGD biopsy can play a significant role affecting DNA quality. These effects are
typically more prevalent in single cell biopsies than trophectoderm, where greater
quantities of starting material are present.
Process for Calling
The fundamentals for calling have been described in previous sections. Overview of
Calling 24sure Single Channel Assays elaborates on previous sections and provides a
summary of the calling process at a high level. The calling of individual changes is then
described in more detail in Calling Individual Changes, with focus on how to deal with
more challenging samples. These figures provide a useful reference for the calling
process.
Summary of Expected X/Y Chromosome Scenarios provides a summary of expected X/Y
chromosome scenarios with images, including common chromosome X and Y disorders,
to assist calling of X/Y chromosomes. In addition, an example of low-level droop on the
Y-chromosome is depicted.
Calling Segmental Imbalances
24sure is not intended for identifying segmental or subchromosomal imbalances. 24sure+
is better equipped for this application. However, in some instances, samples that harbor
segmental changes are clearly apparent in a 24sure assay. The guidelines for calling
segmental changes are the same as whole chromosome changes regarding the tables for
different sample types detailed in this document. However, only a portion of the
chromosome is considered. BlueFuse Multi does not automatically call segmental
changes and 24sure cannot reliably detect imbalances less than 20 Mb in size.
12
Part # 15056973 Rev. A
Low X-Separation
If X-separation is significant relative to the noise of the assay, it is still possible to call
samples with low X-separation. For example, in the blastomere profile in Figure 14, Xseparation is approximately 0.4. Chromosome 2 has a negative log2 ratio directly
comparable to X-separation and therefore corresponds to monosomy.
Figure 14 Blastomere profile with low X-separation, indicates loss on chromosome 2 (red
circle)
Wave and Step Change Effect
The key to calling samples with “wave patterns” is to consider whether the change is of
significant magnitude relative to the X-separation. In the PB2 profile in Figure 15, the
BlueFuse Multi algorithms, which do not exploit the biopsy type, have automatically
called chromosome 13 as a gain. X-separation is high at around 0.9 and a genuine PB2
gain would have a similar magnitude. The 0.3 change on chromosome 13 (red circle) is
clearly insufficient, and is consistent with the background of a noisy sample. This
automated call should be overridden.
Figure 15 PB2 profile with "wave pattern"
Low X-Separation Combined with High Magnitude Wave Effect
The blastomere sample in Figure 16 is challenging to call. However, it is euploid. Most of
the wave effect can be ruled out due to insufficient ratio relative to X-separation. There
are no losses of magnitude similar to the X-separation of 0.4. Genuine gains would be
expected to have a log2 ratio of 0.24. Chromosome 13 is most challenging. Close
inspection shows that only a small proportion of clones has responded at a level
consistent with aneuploidy. Moreover, the change is consistent with the wavy nature of
the background.
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
13
Examples of Calling Challenging Profiles
Examples of Calling Challenging Profiles
Figure 16 Blastomere profile of euploid sample, low X-separation and high magnitude wave
pattern
Example of a Failed Assay
A small proportion of samples generates profiles that cannot be reliably resolved, even
by an experienced operator following all relevant guidelines. From a technical
perspective, these profiles are considered failed assays because they do not provide
strong evidence for aneuploidy or euploidy.
The blastomere profile in Figure 17 illustrates such a scenario. The X-separation is
around 0.6. Monosomies have a similar magnitude, while a trisomy is expected to have
ratio of 0.36. Changes on chromosome 2 and 12 are of insufficient magnitude to be
consistent with genuine deletions. However, the change on chromosome 13 (red circle) is
well-defined, consistent, and of ratio 0.27. The ratio is not quite as high as predicted, and
it is known that this sample contains artifactual step changes on chromosomes 2 and 12.
However, no conclusive call can be made.
Figure 17 A difficult call blastomere profile, example of failed assay
14
Part # 15056973 Rev. A
While calling is straightforward for most profiles, in some cases DNA quality can lead to
profiles that require experience to interpret. The BlueFuse Multi algorithms for automated
calling of 24sure profiles, together with confidence measures provided with each call, are
useful time-saving tools. However, treat the automated calls with caution, particularly
for more complex samples. Automated calls are considered only as an initial
prioritization of potential changes. A trained operator must check automated calls.
It is important to be aware of the limitations of the 24sure Single Channel algorithms in
BlueFuse Multi. The algorithms:
} Only call whole chromosome copy number changes. They do not call segmental
changes, even if clearly present and of significant in size.
} Can over-call small changes that are inconsistent with what is biologically possible.
The algorithms detect changes that appear visually different to the background. They
ignore biopsy type, and therefore do not determine whether changes are of sufficient
log2 ratio to be consistent with genuine biological copy number change.
} Can under-call changes in noisier assays. The algorithms work by assessing the
distance of a proposed change from the noise. If the noise level is high, the
algorithms can sometimes miss a change that is clear to a trained operator.
} Always attempt a call even where the assay has failed. Ignore such calls.
For more complex calls, it is important to rely on the procedures described in this
document rather than automated calls.
When a complex call is made, switch off the green copy number line. With the green
copy number line on, the changes can visually appear more or less real than they
actually are.
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
15
Limitations of Standard Algorithms and the Green Line
Limitations of Standard Algorithms and the Green Line
Overview of Calling 24sure Single Channel Assays
Figure 18 Workflow for calling 24sure Single Channel assays
16
Part # 15056973 Rev. A
Calling Individual Changes
Calling Individual Changes
Figure 19 Workflow for calling individual changes
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
17
Summary of Expected X/Y Chromosome Scenarios
When processed as a Single Channel experiment type, BlueFuse Multi will attempt to
match the sample to the sex of the reference. The matched result is plotted in green, with
the mismatch plotted in pink for a female reference and blue for a male reference. The
mismatched plot provides the valuable X-separation information.
Figure 20 Expected X/Y chromosome scenarios
18
Part # 15056973 Rev. A
For technical assistance, contact Illumina Technical Support.
Table 6 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 7 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on www.cambridgebluegnome.com.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to www.illumina.com/support, select a product, then click Documentation & Literature.
A Technical Guide to Aneuploidy Calling with 24sure Single Channel
Technical Assistance
Technical Assistance
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com