Rapid Aneuploidy and CNV Detection in Single Cells

Technical Note: Reproductive Health
Rapid Aneuploidy and CNV Detection in
Single Cells using the MiSeq® System
Comparison between data generated from single cells using 24sure® array-based screening
and next-generation sequencing (NGS) using the MiSeq System.
Introduction
PGS Using the MiSeq System
IVF success rates are low, with approximately one-third of all cycles
resulting in live births1. Improving IVF success rates would make a
significant difference in the lives of many women trying to conceive.
Chromosome aneuploidy (abnormal number of chromosomes) is a
major cause of IVF failure, miscarriage, and, in rare cases, abnormal
pregnancy and live birth. Most aneuploidies arise during the final
stages of egg development and increase exponentially with maternal
age. Selectively implanting euploid (normal number of chromosomes)
embryos is critical to reducing miscarriage and improving IVF success.
Early clinical experience show improvements as high as 50% in IVF
success rates using embryos selected by the 24sure test2.
Synergy between Leading PGS and NGS Providers
Current Technologies
24sure, launched in 2008, is the method of choice for preimplantation
genetic screening (PGS) and to date over 300,000 clinical samples
have been processed worldwide. This method uses DNA arraybased technology along with state-of-the-art analysis software to
comprehensively and accurately assess all 24 chromosomes.
The 24sure platform is microarray-based, which is a well-established
technology for a wide variety of applications. However, recent
innovations in genomics have led to sequencing-based platforms,
paving the way for improvements in assay workflow and performance.
BlueGnome, acquired by Illumina in 2012, brings together the leader in
preimplantation genetics with the leader in genomics and sequencingbased technology. This synergy benefits from the use of wellestablished protocols, massive libraries of samples for retrospective
studies, and in-depth market knowledge of customer requirements.
Streamlined Sample Management and Common Workflow
The well-established and reliable 24sure assay has been leveraged
in the development of this NGS-based PGS application. An assay
protocol, with a time comparable between both technologies, has
been designed. A common amplification process allows both arrays and sequencing to be conducted on the same amplified products.
This facilitates parallel processing of samples on both platforms for
validation purposes, and the ability to maximize machine availability
and volume benefits.
Shared Interpretation Software
All data can be analyzed in a common consolidated view within
BlueFuse software ensuring a single interpretation workflow across
both technologies. This common approach supports an incremental
transition to sequencing. All sequencing data are processed locally on
the MiSeq System and in BlueFuse Multi so that no server or external
hardware is required.
Figure 1: Common Workflow between Arrays and Sequencing
Sample
Sequencing or Array-Based Screening
Analysis
i
MiSeq system
Amplification kit
Analyze with BlueFuse Multi
24sure
Technical Note: Reproductive Health
Performance Comparison between 24sure and
MiSeq
24sure results were compared with sequencing data generated using
the MiSeq in a proof of principle validation exercise at the BlueGnome
cell culture suite in Cambridge, UK. Single (or multiple cells) from
characterized cultures of known karyotype were isolated into PBS,
then amplified. Individual amplified products were analyzed by 24sure
and sequencing.
For sequencing, these products were purified, quantified, and
concentration normalized. Indexed sequencing libraries were prepared
and normalized using an Illumina Nextera-based method. Library
pools of 12 libraries were sequenced on the MiSeq using 1x36 base
reads. Demultiplexing, alignment, and other secondary analysis was
performed on the MiSeq. BAM files were exported and analyzed in
MatLab®, used as an exploratory tool for BlueFuse Multi development.
Cell Lines
Single (or multiple) cells from characterized cultures of known
karyotype were isolated into PBS, then amplified. Individual amplified
products were analyzed by 24sure and sequencing. Comparison
of data generated from 24sure (Figure 2a) and MiSeq (Figure 2b)
using cells from cultures of known karyotype showed that data was
comparable between the two platforms, demonstrating the feasibility
of using the MiSeq System for PGS.
Single Cell Biopsies from Day 3 Blastomeres
Comparison of data generated from 24sure (Figure 3a) and MiSeq
(Figure 3b) using single cell biopsies from day 3 blastomeres showed
that there was a high level of concordance between the two platforms.
Single Cell Biopsies from Day 5 Trophectoderm
Comparison of data generated from 24sure (Figure 4a) and MiSeq
(Figure 4b) using single cell biopsies from a day 5 trophectoderm
showed that there was a high level of concordance between the two
platforms, even where samples are chaotic.
Figure 2: Comparative Data from a Well-Characterized Cell Line
Data from 24sure (top) and MiSeq (bottom).
Technical Note: Reproductive Health
Figure 3: Comparative Data from a Day 3 Blastomere
Data from 24sure (top) and MiSeq (bottom).
Figure 4: Comparative Data from a Day 5 Trophectoderm
Data from 24sure (top) and MiSeq (bottom).
Technical Note: Reproductive Health
Detection of Segmental Imbalances
Poorer Quality Amplification Products
NGS has the potential to increase the resolution of PGS. Currently,
such information is best interpreted where it is associated with
balanced translocations in the parents. Initial concordance studies
between 24sure (Figure 5a) and MiSeq sequencing data (Figure 5b)
reveal that segmental imbalances are detected by both platforms.
Studies to confirm the exact relationship between resolution and read
depth are ongoing.
One of the most significant challenges of routine PGS is to maximize
the number of samples that are able to be reported. The quality of
the amplification products can vary as a result of biopsy quality or cell
washing, resulting in a noisy array profile. Using NGS on the same
samples, the profiles appear to be cleaner as it is easier to remove
poor quality reads earlier in the analysis pipeline, ensuring that only
high-quality, mapped reads are used to generate the final profile
(Figure 5).
Figure 4: Comparative Data Detecting Segmental Imbalances
Data from 24sure (top) and MiSeq (bottom).
Figure 5: Comparative Profile between Arrays and Sequencing
e
Profile from 24sure (left) and MiSeq (right).
Conclusion
The data confirms that the results are comparable between the arraybased 24sure technology and sequencing using the MiSeq System.
Ongoing validation studies include collaborations with major reference
laboratories in which retrospective analysis of a large number of
samples, previously reported using 24sure, will be undertaken. This
data will be reported when available.
References
1. Fragouli, E. & Wells, D. Aneuploidy screening for embryo selection. Seminars
in Reproductive Medicine 30, 289–301 (2012).
2. Yang, Z. et al. Selection of single blastocysts for fresh transfer via standard
morphology assessment alone and with array CGH for good prognosis IVF
patients: results from a randomized pilot study. Molecular Cytogenetics 5,
24 (2012).
Technical Note: Reproductive Health
Technical Note: Reproductive Health
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Pub. No. 1570-2013-004 Current as of 10 January 2014

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