First Attempt at Complete Sequencing of Blastocyst Biopsies for Use in PGD

Abstract

BACKGROUND: Current techniques that analyze all chromosomal abnormalities in blastocyst biopsies still can only achieve an implantation success rate of approximately 80%. It is possible that this is in part due to array CGH, CGH, SNP arrays, and qPCR not being sensitive enough to detect potential abnormalities associated with implantation failure. The higher resolution of whole genome sequencing could help improve the implantation success rate, as well as provide much more detailed analysis of potential genetic abnormalities. OBJECTIVE(S): To show in a proof-of-principle manner that a complete whole genome sequence can be obtained from a blastocyst and that a known genetic mutation within some of the blastocysts can be identified. MATERIAL AND METHODS: Two DNA amplification METHODS were compared to see which one generates sufficient quality template DNA for whole genome sequence analysis using Complete Genomics' DNA nanoarray sequencing platform (1). The METHODS were SurePlex, which is commonly used for array CGH, and a modified multiple displacement amplification (MDA) (2). 10-20 cells were biopsied from embryos affected with the R-1MT mutation of Myotonic Dystrophy. The samples were lysed and the DNA denatured in a single tube. Approximately 2 ug of DNA were generated by both amplification METHODS. Prior to whole genome sequence analysis, amplified samples were screened with 96 independent qPCR markers spread across the genome to select samples with the lowest amount of bias. Briefly, we determined the average cycle number across the entire plate and subtracted that from each individual marker to compute a “delta cycle” number. The delta cycle was plotted against the GC content of the 1000 base pairs surrounding each marker. This gave us a sense of the relative GC bias of each sample. To get a sense of the overall “noise” of the samples, the absolute value of each delta cycle was summed to create the “sum of deltas” measurement. From our experience, a low sum of deltas and a relatively flat plotting of the data against GC content yields a well-represented whole genome sequence. The best amplified samples were processed by Complete Genomics at their Mountain View, CA sequencing facility as previously described (1). RESULT(S): The sum of deltas was 61 for the MDA method and 287 for the SurePlex. As such, the MDA method was used to amplify the rest of the samples and process them for complete genome sequencing. The results of the sequencing process are expected by the end of December 2010. CONCLUSION(S): Although PGD with whole genome sequencing is currently cost prohibitive, one could amplify DNA, separate an aliquot for analysis by current METHODS, and then sequence the remaining few normal embryos, in order to lower the cost of the procedure. SUPPORT: Departmental funds1.Drmanac et al. (2010) Science 327, 78-81.2.Dean et al. (2002) Proc Natl Acad Sci U S A 99, 5261-5266.