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Application Note

Accurate aneuploidy detection with the Embgenix PGT-A Kit

  • The Embgenix PGT-A Kit is an end-to-end PGT-A solution for next-generation sequencing (NGS)-based preimplantation genetic testing of embryos for aneuploidies (PGT-A). It is offered as research use only (RUO) or CE-marked for in vitro clinical diagnostic use in the European Union.
  • The kit integrates PicoPLEX WGA technology for whole genome amplification (WGA), preparation of a library for Illumina sequencing, and Embgenix Analysis Software for automated data analysis and interpretation. With this kit, clinicians can analyze their genomic DNA (gDNA) and trophectoderm (TE) biopsy samples for accurate determination of aneuploidies.
  • The kit features a streamlined workflow with less hands-on time, the ability to accommodate large input volume, a single WGA dilution factor, and 96 well-plated, dual-indexed, ready-to-use primers for amplification of indexed Illumina-compatible NGS libraries.
  • Embgenix Analysis Software, cloud-based and tailored for automated data interpretation and aneuploidy calling, features Takara Bio’s proprietary copy number variation (CNV) detection algorithm designed for use exclusively with sequencing data generated with the Embgenix PGT-A Kit.
  • The software allows fast and accurate automated calling of aneuploidy including low (30–50%) and high (50–70%) mosaicisms, and sensitivity to detect subchromosomal copy number variations (CNVs) 10 Mb or larger. 

Introduction Results Conclusion Materials and Methods References

Introduction  

The aim of this study was for multiple laboratories—Takara Bio’s in-house laboratory and independent clinical laboratories A, B, and C—to test the performance of the Embgenix PGT-A Kit in detecting whole chromosome aneuploidies, segmental aneuploidies, and mosaicism in TE biopsy samples and gDNA samples.

NGS is a method of choice for PGT-A of in vitro fertilization (IVF) embryos (Vendrell et al. 2017). The overall goal of PGT-A is to improve rates of embryo implantation, live birth, and other metrics of IVF success. Enhanced PGT-A techniques will better inform clinicians providing fertility treatment to patients whose embryos may have complete, partial, or mosaic aneuploidy (Kemper et al. 2020). Common aneuploidies associated with clinical conditions include trisomies of chromosomes 13, 18, and 21 (Chr 13, Chr18, Chr21). With integrated workflows from sample preparation to analysis, NGS-based PGT-A offers a simple and scalable solution for aneuploidy testing in any lab regardless of expertise.

Leveraging our WGA technology, proprietary library prep, and customized bioinformatics pipeline, Embgenix PGT-A is an end-to-end solution for accurately detecting complete, partial, and mosaic aneuploidy in human embryos. The Embgenix PGT-A Kit has been optimized to analyze DNA obtained from embryo biopsies and genomic DNA. The workflow starts with template DNA from the lysis of embryonic cells going through Takara Bio’s PicoPLEX WGA, and then proceeds through our proprietary library preparation using unique dual indexes (UDI). Once purified and quantified, the resulting library is ready for multiplex sequencing on Illumina NGS instruments (Figure 1).

Figure 1.  Embgenix PGT-A workflow. Starting from gDNA, in six to eight hours, any lab can generate libraries in 96-well format ready for sequencing on an Illumina platform.

The PicoPlex WGA process, used to generate enough DNA for NGS library preparation, is shown in Figure 2. This process has a demonstrated superior capacity for amplification with high quality, reproducibility, fidelity, and uniformity (Zhang et al. 2017). Using the WGA product and stem-loop adapters together with UDI primers, our patented library preparation technology prepares a high-quality NGS-ready library (Figure 3). Through sequencing of this library, all 24 chromosomes can be accurately screened for whole-chromosome and subchromosomal aneuploidies.

Figure 2. Takara Bio’s PicoPLEX WGA chemistry. WGA product is generated from gDNA extracted from the TE biopsy sample via cell lysis. A single-tube workflow combines pre-amplification and amplification to generate the WGA product for library preparation.

Figure 3. Takara Bio's patented library preparation. Using stem loop adapters, WGA products are converted to sequencing-ready libraries with Illumina unique dual indexes. Libraries are then ready to be sequenced with Illumina sequencers.

Used after sequencing, Embgenix Analysis Software (Figure 4) is a cloud-based solution allowing analysis of data from single or multiple cell samples from human embryos. With Human Genome number 38 (hg38) as an alignment reference, CNVs are identified and displayed in CNV plots in an idiogram or list format. Users can easily perform CNV analysis via a web-browser, interactively review and amend the CNVs, and generate downloadable PDF reports that can be customized with the users’ own logos.

Figure 4. Embgenix Analysis Software: general workflow for CNV detection and reporting

Results  

Detection of full chromosome and segmental aneuploidies in gDNA samples 

Our first study, using previously characterized gDNA samples, evaluated the feasibility and reliability of the Embgenix PGT-A Kit in detecting full-chromosome and segmental aneuploidies. The Embgenix software detected chromosomal abnormalities affecting whole chromosomes with high accuracy and confidence. An example idiogram resulting from this study is shown in Figure 5.

Figure 5. Embgenix plot showing genomic DNA sample containing trisomy of Chr18.

The Embgenix PGT-A Kit was applied to analyze 22 reference specimens with segmental aneuploidies. Embgenix PGT-A successfully detected 100% of the segmental aneuploidies, which ranged in size from 7.1 Mb to 91.4 Mb, with no false positives called. For 20 of the 22 segmental aneuploidies, Embgenix PGT-A provided highly accurate estimates (within 15%) of CNV size (Table 1). Figure 6 shows example plots of successful predictions by Embgenix PGT-A on clinically significant CNV structures requiring high resolution of segmental detection (Coriell samples NA13019 and NA14485). In some rare cases, the CNV lies across a region of the genome where it is difficult to perform read mapping, the process of aligning the reads on a reference genome. These “low mappability regions” may be trimmed from the reported CNV size, resulting in significant underestimation in the reported CNV size. The underestimation of size for one CNV in Coriell sample NA20556 may arise from this issue, as that CNV extends into a low mappability region.

Table 1. Study with 22 samples, showing accurate reporting of segmental aneuploidies including size prediction.  

Takara Bio’s in-house verification
Coriell Sample Chr True CNV Size (Mb) Embgenix PGT-A Predicted Size (Mb)
NA20022 Chr3 61.1 64
NA14164 Chr13 41.2 48
NA12606 Chr13 41.2 42
NA09367 Chr6 35.2 35
NA11672 Chr10 26.2 26
NA10800 Chr4 25.2 26
NA05966 Chr14 21.2 21
NA11213 Chr2 17.5 18
NA09216 Chr2 16.7 17
NA08331 Chr13 12.1 13
NA10989 Chr9 12 12
NA14943 Chr2 7.8 9
Independent testing at Clinical Laboratory A
Coriell Sample Chr True CNV Size (Mb) Embgenix PGT-A Predicted Size (Mb)
NA13019 ChrX 91.4 98
NA13019 ChrX 56.8 59
NA14485 Chr8 31.1 34
NA06226 Chr16 21.3 22
NA05966 Chr14 21.2 22
NA06870 Chr18 15.4 15
NA20556 Chr15 12.3 7**
NA08331 Chr13 12.1 13
NA16362 Chr22 8* 8
NA14485 Chr8 7.1* 7

*CNV size less than minimum specification for Embgenix Analysis Software. **CNV occurred in region with low mappability.

Figure 6. Analysis using the Embgenix PGT-A Kit demonstrates concordance with previously characterized segmental aneuploidy. Panel A. Embgenix PGT-A correctly predicts two segmental aneuploidies in ChrX, linked to Turner Syndrome, in Coriell gDNA sample NA13019. Panel B. Embgenix PGT-A correctly predicts two segmental aneuploidies in Chr8, linked to hydrocephalus, in Coriell gDNA sample NA14485.

Detection of mosaicism

Next, we tested the accuracy of mosaic detection by Embgenix PGT-A. Two clinical laboratories, A and B, performed studies using mosaics samples created using a mixture of two samples with different aneuploidies. Laboratory A used mixtures of Coriell gDNA samples NA05966 (21.2 Mb trisomy in Chr14) and NA10925 (16.1 Mb monosomy in Chr7). The ratios tested are shown in Figure 7, which presents the results of the study as a comparison between Embgenix PGT-A and another NGS PGT-A method, Reproseq. Embgenix PGT-A successfully detected the segmental mosaicisms represented in the gDNA mixtures at 50% and 75%. Reproseq did not detect the mosaicisms at 50% and 75%.

Figure 7. Comparison of mosaic detection capability between Embgenix PGT-A and Reproseq. Sensitivity of mosaic detection is compared side-by-side for Embgenix PGT-A and Reproseq, using artificial segmental mosaicisms generated by mixing two Coriell gDNA samples (NA05966 and NA10925) containing segmental aneuploidies in different genomic locations. The two Coriell gDNA samples were mixed at the five ratios indicated. CNVs reported by each method are marked as true positives (blue arrows) or false negatives (red squares), with no false positives reported by either method.

The second Clinical Laboratory, B, performed a study using two gDNA samples with known CNVs. The first of these samples contained a 14 Mb segmental monosomy of Chr11. The second gDNA sample contains trisomy of Chr18 (78 Mb). Seven mixtures of these two gDNA samples were prepared. The resulting mixtures were processed using Embgenix PGT-A and Embgenix Analysis Software.

As shown in Table 2, Embgenix PGT-A successfully detected 100% of the 78 Mb trisomy on Chr18 aneuploidy in mosaic mixtures tested. Embgenix PGT-A successfully detected the 14 Mb Chr11 aneuploidy in mosaics where the sample with that aneuploidy made up at least 40% of the mixture.

Table 2. Study of mosaic mixture showing detection of 78 Mb aneuploidy and 14 Mb aneuploidy when the sample with aneuploidy makes up at least 40% of the mixture. 

% mosaicism Chr11, 14 Mb segmental % mosaicism Chr18, full chromosome

Embgenix PGT-A detection: Chr11, 14 Mb segmental

Embgenix PGT-A detection: Chr18, full chromosome
80% 20% Yes Yes
70% 30% Yes Yes
60% 40% Yes Yes
50% 50% Yes Yes
40% 60% Yes Yes
30% 70% No Yes
20% 80% No Yes

Embgenix PGT-A Kit validation using biopsy samples

Multiple IVF clinics collaborated to validate the performance of Embgenix PGT-A with TE biopsy samples. The table of Figure 8, Panel A summarizes two independent performance datasets generated in laboratories at two independent clinics, B and C. In the study performed by Clinical Laboratory B, 34 samples (including 26 TE samples and 8 gDNA samples) were analyzed using Embgenix PGT-A with Embgenix Analysis Software and VeriSeq PGS Kit with BlueFuse Multi Software. Embgenix results were 91% concordant with results obtained from the clinic's established NGS PGT-A analysis method. The three samples generating non-concordant results were from the same embryo. Clinical laboratory B determined these samples probably came from parts of the embryo with different genetic content.

In the study performed by Clinical Laboratory C, multiple biopsies from the trophectoderm (TE) or inner cell mass (ICM) of 71 embryos were analyzed using Embgenix PGT-A and other NGS PGT-A methods and software. Embgenix results were 100% concordant with results obtained from the established NGS PGT-A analysis for each embryo. Figure 8, Panels B, C, and D  show a representative example comparing analysis of two TE samples and one ICM sample from Embryo 9. The results of analysis with Embgenix PGT-A were 100% concordant with data obtained by sample analysis with established methods and software. Figure 9 shows results for another example from this study, Embryo 47.

Clinical Laboratory Sample composition Concordance
B 26 TE biopsies and 8 gDNA samples 91%
C TE or ICM biopsies from 71 embryos 100%

Figure 8. Aneuploidy assessment conducted by Clinical Laboratory C demonstrates concordance between results from Embgenix PGT-A analysis and results from established PGT-A methods. Representative example data from Embryo 9 is shown. Aneuploidy assessment of two TE biopsies and one ICM biopsy were performed using Embgenix PGT-A and an established method, VeriSeq PGS Kit with BlueFuse Multi Software. All indicated monosomy of Chr5. Panel A. Table summarizing Embgenix PGT-A performance on datasets from collaborating IVF clinics. Panel B. Data from a TE sample assessed with VeriSeq PGS Kit with BlueFuse Multi Software. Panel C. Data from a ICM sample assessed with Embgenix PGT-A and Embgenix Analysis Software. Panel D. Data from a TE sample assessed with Embgenix PGT-A and Embgenix Analysis Software.

Figure 9. Aneuploidy assessment conducted by Clinical Laboratory C demonstrates concordance between results from Embgenix PGT-A analysis and results from established PGT-A methods. Representative example data from Embryo 47 is shown. Results from analysis of biopsy samples processed with Embgenix PGT-A and another PGT-A method are in concordance, indicating monosomy of Chr16. Panel A. Data from a TE sample processed with an established NGS PGT-A method and software. Panel B. Data from a TE sample processed with Embgenix PGT-A and Embgenix Analysis Software.

Conclusion  

We have shown that the Embgenix PGT-A Kit, including PicoPlex WGA, preparation of libraries for Illumina sequencing, and Embgenix Analysis Software genomic data analysis and aneuploidy screening, is a complete solution for PGT-A.

Data demonstrate Embgenix PGT-A rapidly and accurately calls whole-chromosome aneuploidies, segmental chromosomal aneuploidies > 10 Mb, as well as low (30-50%) and high (50-70%) mosaicisms in defined mixtures of samples with known aneuploidies.

Results also show Embgenix PGT-A accurately detects the size of CNVs in segmental aneuploidies affecting an unbiased selection of chromosomal regions. Testing in independent clinical laboratories has shown concordance between results from Embgenix PGT-A and results from established methods of NGS PGT-A. This testing used both gDNA and TE and ICM biopsies.

Taken together, results clearly show the feasibility of clinical application of Embgenix PGT-A and demonstrate the kit as a viable solution for the needs of current and future PGT-A.

Materials and Methods  

Sample collection

For experiments performed by Takara Bio's in-house laboratory, cell lines with defined segmental aneuploidies were purchased from Coriell Biorepository (USA) as reported in Table 3. 26 clinical TE biopsies were obtained by Clinical Laboratory B. A total of 71 embryos were included in the study conducted by Clinical Laboratory C. For each embryo, multiple biopsies were taken from the trophectoderm (TE) or inner cell mass (ICM).  

Table 3. Panel of Coriell gDNA samples containing segmental aneuploidies

Coriell ID Description Size (Mb)
NA05966 Duplicated chromosome 21.2
NA06226  Translocated chromosome 21.3
NA06870  Aneuploid chromosome number - non-trisomic 15.4
NA08331  Chromosome deletion 12.1
NA09216  Chromosome deletion 16.7
NA09367  Duplicated chromosome 35.2
NA10800  Chromosome deletion 25.2
NA10989

Gilles de la Tourette syndrome (GTS)

Chromosome deletion

12.0
NA11213  Chromosome deletion 17.5
NA11672  Chromosome deletion 26.2
NA12606  Aneuploid chromosome number - non-trisomic 41.2
NA13019 

Chromosome deletion

Turner syndrome

91.4, 56.8
NA14164

Tetralogy of Fallot

Chromosome deletion

41.2
NA14485 

Inverted duplication deletion

7.1, 31.1
NA14943  Chromosome deletion 7.8
NA16362  Aneuploid chromosome number - trisomy 8.0
NA20022  Duplicated chromosome 61.1
NA20556  Isodicentric chromosome 12.3

Whole genome amplification

Samples were processed following the Embgenix PGT-A protocol. The PicoPlex WGA Kit uses a single-tube protocol for the amplification of genomic DNA starting from samples stored in liquid N2, a standard practice for WGA starting material in this field.

Sequencing

Sequencing-ready libraries were pooled and sequenced using MiSeq® with 24 samples per flow cell or using NextSeq® with 96 samples per flow cell.

Genomic data analysis and aneuploidy screening

Sequencing data were analyzed using the Embgenix Analysis Software.

References  

Kemper, J. M. et al. Preimplantation Genetic Testing for Aneuploidy: A Review. Obstet Gynecol Survey 74(12), 727–737 (2019).

Vendrell, X. et al. New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos. Systems Biology in Reproductive Medicine 63(3), 162–178 (2017).

Zhang, X. et al. The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection. Bioscience Reports 37 BSR20170252 (2017)

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