The Agilent GenetiSure Pre-Screen Kit with Rubicon PicoPLEX WGA for Pre-implantation Genetic Screening by Array CGH

Product Note

The Agilent GenetiSure Pre-Screen Kit is a cost-effective solution that enables:

• Rapid identification of aneuploidies of embryonic biopsied cells

• Detection of genomic aberrations down to 10 Mb and as low as 5 Mb*

• Sample-to-results in less than 24 hours

• Cost-effective screening of up to 14 samples per slide

• Kit includes male and female reference DNA

• An intuitive user interface for single cell triage and easily generated analysis reports with Agilent CytoGenomics software

* Using the Single Cell Small Aberration Method

High resolution and cost-effective genetic screening of embryonic cells prior to implantationGenomic profiling a small number of cells is very productive in in vitro fertilization research. The power of genome-wide copy number profiling obtained with the Agilent oligonucleotide microarray CGH platform exceeds the limited resolution of traditional techniques such as FISH, PCR, and BAC arrays.

The Agilent GenetiSure Pre-Screen workflow combines whole genome amplification using the Rubicon PicoPLEX amplification method with oligonucleotide microarrays specifically designed for embryonic biopsied cell screening. Agilent provides a comprehensive array, reagent, and software solution for screening prior to transfer for implantation.

The power of Agilent aCGH with Oligonucleotide Library Synthesis (OLS) TechnologyArray comparative genomic hybridization (aCGH) analysis has revolutionized the field of cytogenetics by providing accurate identification of copy number variations. Agilent oligo-based arrays provide higher resolution and reproducibility than bacterial artificial chromosome (BAC) arrays. This is because Agilent arrays have 60,000 high fidelity probes as compared to the 3,000 probes of BAC arrays. Agilent high resolution arrays can fit 14 samples per slide, and offer a cost-effective solution for preimplantation genetics screening (PGS).

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A superior platform for prescreening of biopsied embryonic cellsPre-implantation genetics research is a fast growing field driven by technological advances that enable researchers to investigate aneuploidies in a small number of embryonic cells. Conventional single cell analysis methods, including fluorescence in situ hybridization (FISH), lack genome-wide scalability.

In response to this limitation, high-throughput aCGH technology has become increasingly valuable in assessing genome-wide copy number (CN) in embryonic biopsy samples. Bacterial artificial chromosome (BAC) arrays offer genome-wide CN detection but suffer from low resolution due to shorter probe length and the availability of only a few thousand probes on the array.

Agilent high-resolution and highly sensitive oligonucleotide aCGH technology provides the required resolution to detect CN changes down to 5 Mb, using only a few embryonic cells. The GenetiSure Pre-Screen Kit with Rubicon PicoPLEX includes 8x60K format microarrays, the SureTag labeling kit, and the Rubicon Genomics Whole Genome Amplification (WGA) Kit. The unique microarray format enables the simultaneous analysis of up to 14 tested samples plus male and female reference genomic DNA. The 60,000 probes were optimized to provide genome-wide coverage, with increased density on chromosomes 13, 18, 20, 21, 22, and X, which are commonly associated with aneuploidy events (Figure 1).

The PicoPLEX WGA kit with patented technology is designed and optimized for amplification of a single cell copy genomic DNA (Figure 2). The easy-to-use single tube protocol reduces handling errors, dramatically improves time to results, and reduces background noise.

Figure 1. Genome-wide probe coverage, with increased density on chromosomes 13, 18, 20, 21, 22, X and Y.

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Agilent SurePrint G3 Human CGH 8x60KAgilent GenetiSure Pre-Screen 8x60K

Figure 2. PCR-based whole genome amplification with Rubicon Genomics PicoPLEX WGA. A three-step, single-tube reaction starts with a few cells. Cellular genomic DNA (gDNA) extracted in step 1 was used as a template for multiple cycles of quasi-random priming and linear amplification followed by exponential library amplification. The GenetiSure Pre-Screen protocol recommends using 3–5 cells.

Single cell

Step 1: Cell lysis

Step 3: Amplification

Step 2: Preamp(12 cycles)

Template DNA

Denature torecycle template

Accumulatinghairpin library

Extend

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Extend primers

Anneal primers

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Agilent GenetiSure Pre-Screen Array 8x60K• Up to 14 biological samples can be

tested on one slide (Figure 3)

• Samples are differentially labeled with Cy3 and Cy5, and combined in pairs for single-channel analysis

• Reference male and female gDNA samples are also amplified and processed in parallel with biological samples

High-resolution detection of copy number aberrations for pre-implantation screeningFigure 4 illustrates the detection of chromosomal aberrations in the model system cell lines (3–5 cells) from Coriell ranging from trisomy of an entire chromosome down to a 5.3 Mb loss. Using the Single Cell Recommended Analysis Method in CytoGenomics software, the detection rate was 100 % for aberrations >10 Mb, and 89 % for > 5.3 Mb across six cell lines (44 samples from each cell line, total 264 tested samples). When the Single Cell Small Aberration Analysis Method was used for samples with known aberration of 5.3 Mb in cell line GM09189, it was successfully detected in all 44 tested samples, improving the detection rate to 100 %.

Using 5 to 15 cells from day 5 embryos, several aberrations were detected that were also tested using next-generation sequencing (NGS). Figure 5 illustrates losses detected in the embryos of chromosomes 18, 22, and 13, as well as gains of chromosomes 7, 11, and 19. All of these aneuploidies were confirmed using NGS.

Agilent

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Figure 3. Schematic sample layout of an Agilent GenetiSure 8x60K array.

Figure 4. Whole-genome CN profiling of different known aberrations across six different Coriell cell lines, with detection down to 5.3 Mb.

GM02732

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Chromosome 18 aneuploidy

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High-resolution, lower cost pre-implantation screening Agilent oligonucleotide aCGH technology enables the resolution of gross genomic abnormalities down to 5 Mb. With a lower cost per sample than conventional or competitive methods, more embryos can be screened without significantly increasing costs. Easy-to-use arrays integrate the GenetiSure Pre-Screen Kit into a lab’s workflow. Sample-to-results is complete within 24 hours, allowing quick prioritization of embryos for implantation.

Agilent GenetiSure Pre-Screen arrayA B Confirmed by NGS

Abnormal (–18, –22)

Abnormal (–13)

Abnormal (+11)

Abnormal (+7, +19)

Figure 5. Four samples from day 5 embryos (5–15 cells) analyzed using the Agilent GenetiSure Pre-Screen Kit with Rubicon PicoPLEX (A), and confirmed with NGS. Data was contributed by PacGenomics, California, USA.

Complete CGH array workflowThe GenetiSure Pre-Screen Kit with Rubicon PicoPLEX contains all the amplification and labeling reagents, reference samples, and aCGH arrays required to run your samples. There is enough capacity per glass slide to run up to 14 test and two reference samples simultaneously. This kit provides maximum throughput and efficiency for your pre-implantation screening workflow.

Agilent also provides an end-to-end solution that includes hybridization and wash solutions, a hybridization oven, SureScan microarray scanner, and Agilent CytoGenomics software for easy data analysis (Figure 6).

Figure 6. The complete Agilent GenetiSure Pre-Screen workflow.

Sample preparation 15 minutes

180 minutes

135 minutes

60 minutes

35 minutes

16 hours

45 minutes

Total ~24 hours

Whole genome amplification

Labeling

Purification

Preparation for hybridization

Hybridization

Washing, scanning, and analysis

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Agilent GenetiSure Pre-Screen Kit w/ Rubicon PicoPLEX (p/n G5979A)Agilent GenetiSure Pre-Screen Array Kit 8x60K, 3 slides (p/n G5963A)Rubicon Genomics R30050; PicoPLEX WGA Kit, 50 reactions (p/n 5190-9533)SureTag Complete DNA Labeling Kit (w/Agilent M/F Reference, 100 reactions) (p/n 5190-4240)Hybridization Chamber Gasket Slide Kit 8-pack, 3 gasket slides (p/n G2534-60018)

Additional componentsHuman Cot-1 DNA p/n 5190-3393Agilent Oligo aCGH Hybridization Kit (25) or (100) 5188-5220 or 5188-5380Agilent Oligo aCGH Wash Buffer 1 and 2 Set 5188-5226Hybridization chamber, stainless G2534AHybridization oven G2545AHybridization oven rotator rack G2530-60029SureScan microarray scanner* G4900DASureTag purification columns† 5190-3391Agilent CytoGenomics software v4.0 license (free)

Technical specifications: Agilent GenetiSure Pre-Screen Array Kit 8x60K, 3 slides (p/n G5963A)Format 8x60KDesign ID 067559Total features 62,976Control grid feature count 3886Biological features 55,090Distinct biological features 54,911Median probe spacing 49,763Average probe spacing 52,106Chromosomes with increased density 13,18, 20, 21, 22, X, and Y

* G5761AA SureScan Dx available in select countries.† The SureTag Complete DNA Labeling Kit includes 50 SureTag purification columns. Use p/n 5190-3391

to purchase additional columns.

Ordering InformationEach kit can process 48 reactions.

www.agilent.com/genomics

For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

PR7000-0279

© Agilent Technologies, Inc., 2017 Published in the USA, January 5, 2017 5991-7447EN

PicoPLEX® WGA Kit is a registered product of Rubicon Genomics