The WGA reaction (Figure 1) starts with cell lysis, and then DNA is used as a template for preamplification using a quasi-linear amplification approach. This ensures the DNA template is reprimed and amplified in the same manner to create a library of hairpin molecules that cannot be further amplified. These hairpin molecules are a key intermediate ensuring that all amplified molecules are copies of the original DNA template rather than copies of copies. This approach ensures that low-level errors that may be introduced during PCR are not amplified, reducing false signals in the final libraries. These hairpins, in turn, are directly amplified into bulk quantities of DNA used for downstream PGT applications. A highly optimized change in the amplification conditions releases the hairpins to be further amplified in bulk with universal primers, leading to a high-complexity mixture of DNA molecules with minimal amplification bias.
Preimplantation genetic testing powered by PicoPLEX technology
Assisted reproductive technologies provide tremendous hope for couples who are trying to start or grow their families. These couples often struggle with infertility or have a family history of a serious disease and worry about passing it on to their offspring. In vitro fertilization (IVF) is one procedure used to treat infertility, and a couple may opt to select healthy embryos before implantation to help ensure a successful pregnancy. In this case, preimplantation genetic testing (PGT) is performed to screen embryos for chromosomal abnormalities (PGT for aneuploidy [PGT-A]) or known/suspected genetic conditions (PGT for monogenic diseases [PGT-M]). Advances in PGT techniques and technologies have made a real difference in increasing the chances of pregnancy and reducing the risk of miscarriage and congenital disabilities due to genetic disorders. PGT can also shorten the time to pregnancy and lower costs by allowing the prioritization of embryos for implantation, thereby reducing the total number of IVF cycles.
Samples for PGT are typically obtained by taking a biopsy of a few cells, referred to as a trophectoderm biopsy. Although a variety of PGT methods have been used in the past (such as fluorescence in situ hybridization [FISH] and array comparative genomic hybridization [aCGH] assays), next-generation sequencing (NGS) is currently preferred as the most advanced and comprehensive PGT method. NGS-based PGT involves extracting DNA from the trophectoderm biopsy, amplifying the DNA to appropriate input levels for NGS library prep, sequencing, and analyzing data to examine chromosome structure and number. This allows the determination of whether an embryo is euploid, aneuploid, mosaic, and/or carrying single-gene mutations. This information enables the physician to select an embryo for transfer that has the greatest chance of success.
The majority of PGT workflows have incorporated Takara Bio's PicoPLEX whole genome amplification (WGA) technology into their testing protocols. PicoPLEX technology is designed and optimized for unbiased amplification of single-copy genomic DNA from inputs as low as a single cell or cell-free DNA from liquid biopsies. The simple protocol reduces handling errors, dramatically improves time to results, and reduces background. It is trusted as the gold-standard DNA amplification technology for PGT due to its accuracy and reproducibility.
PicoPLEX technology: how does it work?
PicoPLEX inside: NGS-based PGT
The following publications demonstrate NGS-based PGT-A utilizing PicoPLEX technology to improve embryo screening outcomes, understand mosaicism (which can significantly complicate diagnoses), and discover changes in gene expression during embryo development.
Vendrell, X. et al. New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos. Syst. Biol. Reprod. Med. 63, 162–178 (2017).
The authors developed a highly reliable assay for detecting aneuploidy in single blastomeres and samples of 5–10 trophectoderm cells. Their approach—a low-coverage whole genome sequencing method from WGA libraries plus an original algorithm and copy number viewer—depended on PicoPLEX technology to amplify nanogram amounts of gDNA from picogram-level inputs for their NGS protocol. They combined PicoPLEX's limited displacement preamplification with barcodes, followed by PCR amplification, and achieved both a high level of useful reads as well as full correlation of chromosome dose with samples processed using a CGH-BAC microarray approach. They found no differences in sequencing and mapping parameters between unamplified control DNA samples and WGA products from single blastomeres, indicating an unbiased coverage using PicoPLEX technology. Their assay enabled the accurate detection of segmental aneuploidy, with rearrangements of segments down to 1 Mb in length.
Vera-Rodríguez, M. et al. Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing. Fertil. Steril. 105, 1047–1055.e2 (2016).
The authors evaluated a commonly used NGS-based PGT-A technique for the detection of segmental and whole-chromosome aneuploidies from trophectoderm biopsies and compared these results to the typical aCGH approach. The overall concordance rate between NGS and aCGH was 99.8%, and 92.9% of segments detected by both NGS and aCGH were confirmed by FISH. PicoPLEX technology was used for whole genome amplification, followed by library preparation. The NGS approach detected segmental aneuploidies down to 10 Mb with the same efficiency as aCGH; furthermore, NGS detected several mosaic segmental aneuploidies that were not detected by aCGH. Additionally, the authors determined that most of the segmental aneuploidies resulted from mitotic errors leading to mosaicism, and this mosaic pattern was reliably detected by NGS.
Macaulay, I. C. et al. G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015).
The authors introduced a new technique, genome and transcriptome sequencing (G&T-seq), which enables a more in-depth characterization of single cells through simultaneous full-length RNA sequencing, whole-genome or targeted sequencing, and genome-wide copy number variation (CNV) detection. Their method involves bead-based isolation of polyA+ mRNA from gDNA, and then separate amplification, library preparation, and sequencing. PicoPLEX technology was chosen to amplify DNA for downstream CNV analysis. The authors produced several interesting and novel findings using their G&T-seq method: 1) chromosomal copy number in a single cell is corroborated by the expected changes in gene expression in that cell; 2) an aneuploidy event that occurs during a single cell division tracks with gene expression dosage; 3) within a single cell, there is high concordance of single nucleotide variation detected in gDNA and mRNA; and 4) a previously unknown fusion and the causative chromosomal rearrangement underlying the fusion were discovered.
Fuchs Weizman, N. et al. Towards improving embryo prioritization: parallel next generation sequencing of DNA and RNA from a single trophectoderm biopsy. Sci. Rep. 9, 2853 (2019).
The authors created a clinically applicable method, called preimplantation genetic testing for aneuploidy and transcriptome (PGT-AT), that prepares a biopsy of 4–6 trophectoderm cells for simultaneous DNA- and RNA-seq to determine the ploidy status and transcriptome profile. In this study, two gold-standard Takara Bio technologies were employed: SMART-Seq chemistry for cDNA library preparation from full-length mRNA and PicoPLEX chemistry for cell lysis, whole genome amplification, and library preparation. In addition to achieving 100% concordance of CNV detection between the standard PGT-A method and the PGT-AT method performed on the same blastocysts, they discovered that pathways regulating gene expression and energy metabolism were downregulated in the euploid samples. The authors expressed confidence that the transcriptome profiling aspect could be safely introduced into the current clinical PGT-A workflow, and doing so could greatly improve our embryo prioritization abilities.
Tortoriello, D. V., Dayal, M., Beyhan, Z., Yakut, T. & Keskintepe, L. Reanalysis of human blastocysts with different molecular genetic screening platforms reveals significant discordance in ploidy status. J. Assist. Reprod. Genet. 33, 1467–1471 (2016).
The authors reanalyzed blastocysts that returned abnormal PGT-A results to determine the reliability of different assays run by different labs. They examined the levels of concordance between the first and second rounds of trophectoderm biopsies analyzed by two different PGS labs using aCGH or SNP array (first biopsy/lab) and SNP array or NGS (second biopsy/lab). For NGS, WGA was performed with PicoPLEX technology. Chaotic results were obtained, including normal karyotype or gender-discrepant results upon second biopsy, and it could not be determined whether the lack of concordance was due to mosaicism, differences between platforms, or human error. The authors recommended regular, independent oversight verifying test performance and accuracy.
Screen CNV and SNV in a single tube
Takara Bio attended PGDIS 2019 to present our novel single-tube assay that enabled the simultaneous detection of both copy number variation (CNV) across the whole genome and single-nucleotide variation (SNV) or small insertion/deletions (indels) located in the CFTR gene target region.View data
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