Next generation sequencing for preimplantation genetic diagnosis

19 02 2013

To the Editor:

We read with interest the editorial written by Simpson and colleagues regarding next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) (1). While the authors provide their debatable opinion regarding the history, success, and failure of prior and existing methodologies for genetic analysis of the human embryo, here we will focus only on the major concern raised over allele dropout (ADO) after using NGS, and the proposed limitations of NGS in PGD, which are based upon inaccurate assumptions of the methodology and a misrepresentation of contemporary PGD.

First, the authors incorrectly suggest that NGS-based PGD cannot assess trinucleotide repeat disorders such as Fragile X syndrome. For instance, some NGS methods with longer read length capability (2) may provide enhanced ability and more accurate direct analysis of repeat size and expansion (3). Also, given that these types of disorders are often diagnosed indirectly through the evaluation of linked informative polymorphisms and not directly through repeat size analysis, NGS is even more capable than current methodologies. Specifically, NGS provides an opportunity for increased parallel analysis of multiple markers, and a unique opportunity for direct phasing (4), to predict inheritance of mutations and to reduce the risk of misdiagnosis from ADO.

Second, the authors inaccurately suggest that, “whole genome amplification (WGA), using universal primers, must first be performed” for NGS analysis of the embryo [emphasis added]. In fact, our recent paper specifically did not use WGA to perform NGS for PGD (5). Rather, a targeted approach with sequence specific primers was employed. Therefore, the data regarding ADO after WGA is not applicable to predicting the rates of ADO by the specific methodology of NGS described in our study and perhaps many future studies involving NGS for PGD. The confusion may stem from the recent publications on single cell whole genome NGS for aneuploidy detection, which have involved a variety of WGA methods (6-9). While WGA may be appropriate for aneuploidy screening by NGS, we agree with Simpson and colleagues that it is not appropriate for single gene disorder screening, which is why we did not use WGA in our study (5).

Third, the authors inappropriately use single-cell ADO rates to represent expectations from contemporary PGD. This contradicts their own statement that “trophectoderm (blastocyst) rather than cleavage-stage (blastomere) biopsy is increasingly being used.” It has been demonstrated that the rates of ADO and failure to recover all genomic sequences are significantly higher from a single blastomere than from a trophectoderm biopsy where multiple cells are obtained (10). In our own experience, the magnitude of increase in ADO rates from single cells such as a blastomere is tenfold relative to ADO rates from multiple cells such as a trophectoderm biopsy (11). The difference in performance is even further magnified when the reliability of obtaining a result is also considered, an important omission from Simpson and colleagues. For example, single blastomere biopsies have been found to give a genetic diagnosis 12%-19% less often than trophectoderm biopsies (10), including results obtained from the Reproductive Genetics Institute (12). This is certainly one reason why blastomere biopsy, with any type of genetic testing, may soon be considered outside the standard of care. Our NGS study specifically evaluated a protocol for trophectoderm biopsies (5) such that single-cell ADO rates are not applicable, nor are they applicable to future studies that apply NGS on trophectoderm biopsies. Given the clear trend toward increasing use of trophectoderm biopsy as an alternative to blastomere biopsy, in large part due to the negative impact of the procedure (13), and improved precision and reliability of genetic analyses, it is misleading to utilize data on single cells to represent performance expectations of contemporary PGD technologies.

Clearly, ADO is and always has been an important consideration for methods of monogenetic disease analysis in human embryos, and while NGS-based analysis of trophectoderm biopsies may not completely overcome the associated risks, it should not be considered any more prone than existing methodologies. As new technologies become available, it is just as important to avoid inappropriate assumptions regarding methodological limitations as it is to consider legitimate concerns. The real focus of future work involving NGS for PGD is therefore not based on a newfound concern over ADO, but instead on identifying the necessary depth and coverage to maintain sufficient accuracy, and to continue to explore the vast opportunities that this breakthrough technology now provides.

Nathan R. Treff, Ph.D.
Eric J Forman, M.D.
Richard T. Scott Jr., M.D.
Reproductive Medicine Associates of New Jersey


1. Simpson JL, Rechitsky S, Kuliev A. Next generation sequencing for preimplantation genetic diagnosis. Fertil Steril 2013; In press.

2. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, et al. Real-time DNA sequencing from single polymerase molecules. Science 2009;323:133-8.

3. Loomis EW, Eid JS, Peluso P, Yin J, Hickey L, Rank D, et al. Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res 2013;23:121-8.

4. Kitzman JO, Mackenzie AP, Adey A, Hiatt JB, Patwardhan RP, Sudmant PH, et al. Haplotype-resolved genome sequencing of a Gujarati Indian individual. Nat Biotechnol 2011;29:59-63.

5. Treff NR, Fedick A, Tao X, Devkota B, Taylor D, Scott Jr. RT. Evaluation of targeted next-generation sequencing-based preimplantation genetic diagnosis of monogenic disease. Fertil Steril 2013. In press.

6. Yin X, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, et al. Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts. Biol Reprod 2013.

7. Zhang C, Zhang C, Chen S, Yin X, Pan X, Lin G, et al. A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing. PloS One 2013;8:e54236.

8. Baslan T, Kendall J, Rodgers L, Cox H, Riggs M, Stepansky A, et al. Genome-wide copy number analysis of single cells. Nat Protoc 2012;7:1024-41.

9. Lu S, Zong C, Fan W, Yang M, Li J, Chapman AR, et al. Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science 2012;338:1627-30.

10. Kokkali G, Traeger-Synodinos J, Vrettou C, Stavrou D, Jones GM, Cram DS, et al. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study. Hum Reprod 2007;22:1443-9.

11. Tao X, Su J, Pepe R, Northrop LE, Ferry KM, Treff NR. PGD for monogenic disease by direct mutation analysis alone in 2 or more cells is more reliable than multiple marker analysis in single cells. Fertil Steril 2011;96:S21.

12. Forman EJ, Ferry KM, Gueye N-A, Smith RD, Stevens J, Scott Jr RT. Trophectoderm biopsy for single-gene disorder preimplantation genetic diagnosis (PGD) is significantly more reliable than day 3 blastomere biopsy. Fertil Steril 2011;96:S222.

13. Treff NR, Ferry KM, Zhao T, Su J, Forman EJ, Scott RT. Cleavage stage embryo biopsy significantly impairs embryonic reproductive potential while blastocyst biopsy does not: a novel paired analysis of cotransferred biopsied and non-biopsied sibling embryos. Fertil Steril 2011;96:S2.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2012.02.034




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