Using combined RNA/DNA short read sequencing to investigate allele-specific expression from the inactive X chromosome in human cells

Many genomic regions exhibit allele-specific expression. This effect is most pronounced in imprinted genes, where one copy of a gene is epigenetically silenced, and the inactive X chromosome of female cells, where almost the entire chromosome is silenced. Allele specific gene expression can have significant effects on human health and is implicated in a wide array of diseases. Research into allele specific expression is most often carried out in mouse models where cross breeding of mouse strains can yield progeny with well characterised haplotypes where parent of origin is known for a huge number of SNPs. The same approach cannot be taken with human data and haplotypes must be assembled using expensive and labour intensive long read sequencing and Hi-C based approaches. Although resolved haplotypes are available for a number of cell lines, allowing accurate measurement of allele-specific gene expression, this type of analysis is inaccessible for non-specialist labs. We demonstrate how to use previously published haplotypes to investigate X linked gene silencing and epigenetic changes. Additionally, in this paper we present a method to exploit the profound difference in expression levels between the two human X chromosomes to assign SNPs in expressed RNA to the active or inactive X chromosome using only short read DNA and RNA sequencing. We demonstrate this technique using sequencing libraries generated in house and sequencing data from publicly available databases including for a cell line with a complex karyotype. In each instance we identified genes that were silenced in each cell line opening them up to further research avenues. This X chromosome haplotyping technique can be applied to any clonally derived human cell line with 2 or more X chromosomes allowing researchers to investigate X linked gene silencing in cell lines already present in their lab rather than in the limited number of cell lines for which a haplotype is available.