Rapid Aneuploidy and CNV Detection in Single Cells using the MiSeq System

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1 i 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 IVF success rates are low, with approximately one-third of all cycles resulting in live births 1. 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 test 2. 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. PGS Using the MiSeq System Synergy between Leading PGS and NGS Providers 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 MiSeq system Amplification kit Analyze with BlueFuse Multi 24sure

2 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

3 Figure 3: Comparative Data from a Day 3 Blastomere Figure 4: Comparative Data from a Day 5 Trophectoderm

4 Detection of Segmental Imbalances 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. Poorer Quality Amplification Products 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 Figure 5: Comparative Profile between Arrays and Sequencing e Profile from 24sure (left) and MiSeq (right).

5 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, (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).

6 Illumina toll-free (U.S.) tel FOR INTERNAL USE ONLY 2014 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cbot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iselect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No Current as of 10 January 2014