Supplementary code to: Sexing of cattle embryos using RNA-sequencing data or polymerase chain reaction based on the completed sequence of cattle chromosome Y

Background: When necessary, RNA-sequencing data or polymerase chain reaction (PCR) assays can determine the presence of chromosome Y (ChrY) in samples so that biological variation due to sexual dimorphism can be studied. A prime example is when researchers conduct RNA-sequencing of single embryos, or conceptuses prior to the development of gonads. A recent publication of a complete sequence of ChrY has removed limitations for the development of these procedures in cattle, otherwise imposed by the absence of a ChrY in the reference genome. Using the sequence of the cattle ChrY and transcriptome data, we conducted a systematic search for genes in the ChrY that are exclusively expressed in male tissues.

Results: The genes ENSBIXG00000029763, ENSBIXG00000029774, ENSBIXG00000029788, and ENSBIXG00000029892 were consistently expressed across male tissues and lowly expressed or absent in female samples. We observed that the cumulative values of counts per million were 2688-fold greater in males than the equivalent values in female samples. Thus, we deemed these genes suitable for the sexing of samples using RNA-sequencing data. We successfully used this set of genes to infer the sex of 22 cattle blastocysts (eight females and fourteen males). Additionally, the completed sequence of the cattle ChrY has segments in the male-specific region that are not repeated, and we designed a pair of oligonucleotides that targets one of the non-repeated regions in the male-specific sequence of the ChrY. Using this pair of oligonucleotides, in a multiplexed PCR assay with oligonucleotides that anneal to an autosome chromosome, we accurately identified the sex of cattle blastocysts.

Conclusions: We developed efficient procedures for the sexing of samples in cattle using either transcriptome data or their DNA. The procedures using RNA-sequencing will greatly benefit researchers who work with samples limited in cell numbers, only sufficient to produce transcriptome data. The oligonucleotides used for the accurate sexing of samples using PCR are transferable to other cattle samples.