Determination of single embryo sex in Macaca mulatta and Mus musculus RNA-Seq transcriptome profiles

Abstract

To account for sex as a biological variable, it is sometimes necessary to identify the sex of an embryo or embryonic cell that was used to generate libraries for RNA sequencing, without the sex being known a priori. The preferred approach for this would take advantage of the mRNA data, rather than relying on other methods that require separation and analysis of genomic DNA or diversion of limiting RNA for other assays. We describe here a method that has been optimized for this purpose in samples of rhesus monkey and mouse embryos. This method is broadly applicable to any species for which a sufficiently well characterized genome and knowledge of polymorphisms are available, and for embryos that are transcriptionally active and expressing their genome.

Keywords: RNAseq; embryo sexing; nonhuman primate.

Fig. 1.

Summary of method for determining sex of embryos or cells used to make RNA-Seq libraries. A: the three-step process and decision-making tree for determining the sex of embryos from which RNA-Seq libraries were derived. B: scatter-plot of marker expression values, with visual representation of regions corresponding to three steps: embryos identified as male (step #1), embryos identified as female (step #2), and inconclusive (step #3 needs to be applied). SNP, single nucleotide polymorphism; FPKM, fragments per kilobase of transcript per million mapped reads.

Fig. 2.

Scatter-plot of female (Xist) vs. male (Eif2s3y) marker expression measured by RNA-Seq for wild-type (WT) (A) and Xist WT/knockout (KO) (B) mouse embryos in GSE80810. Sex of embryos was determined by measuring expression of same markers with PCR assay (o, female; x, male). Insets: areas close to the origin shown in more detail, where embryos with strong expression of Xist or Eif2s3y are marked as female (F) or male (M), for the remaining embryos the expression-based method is inconclusive. These embryos are listed in the table at the bottom of the figure, which shows expression of Xist and Eif2s3y, as well as the percentage of X-chromosome SNPs with observed biallelic expression, which is used to resolve ambiguity (>5% – female, <5% – male). Emb, embryo; RPRT, reads per retro-transcribed length per million mapped reads. The “?” entries denote embryos unresolved for sex by RNA-Seq due to a high percentage of SNPs with HetScore ≥ 0.75 plus high expression of Eif2s3y. Embryo types are two-cell (2C), eight-cell (8C), 16-cell (16C), 32-cell (32C), and blastocyst (BLA) stages.