Using next-generation RNA sequencing to identify imprinted genes

Abstract

Genomic imprinting is manifested as differential allelic expression (DAE) depending on the parent-of-origin. The most direct way to identify imprinted genes is to directly score the DAE in a context where one can identify which parent transmitted each allele. Because many genes display DAE, simply scoring DAE in an individual is not sufficient to identify imprinted genes. In this paper, we outline many technical aspects of a scheme for identification of imprinted genes that makes use of RNA sequencing (RNA-seq) from tissues isolated from F1 offspring derived from the pair of reciprocal crosses. Ideally, the parental lines are from two inbred strains that are not closely related to each other. Aspects of tissue purity, RNA extraction, library preparation and bioinformatic inference of imprinting are all covered. These methods have already been applied in a number of organisms, and one of the most striking results is the evolutionary fluidity with which novel imprinted genes are gained and lost within genomes. The general methodology is also applicable to a wide range of other biological problems that require quantification of allele-specific expression using RNA-seq, such as cis-regulation of gene expression, X chromosome inactivation and random monoallelic expression.

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

“…They are essential in determining the origin of a parental allele of genes. However, mapping at SNP locations can introduce alignment bias towards the reference alleles because the reads of the alternative alleles may be treated as mismatches and discarded by the mapping tool [7]. To minimize such bias, we artificially built a pseudo-genome (named ‘alternative genome’) by flipping the reference/alternative alleles at all SNP sites from the dbSNP database (sheep 9940) of the sheep reference genome.…”

“…Among those challenges are alignment bias of RNA-seq reads and filtering potential false positive SNPs. Heterozygosity can increase mapping bias because a read from the non-reference allele is considered a mismatch, resulting in a low mapping rate [7]. To minimize such alignment bias to the reference allele in the genome, we artificially built a pseudo-genome (named ‘alternative genome’) by flipping the reference/alternative alleles in all SNP sites based on known sheep dbSNP (sheep 9940).…”

“…This is because low read coverage may have a large variance in differential allelic expression estimation, potentially generating false positive differences [7]. We then identified the expressed parental allele of all SNPs in the same gene and removed genes which contained discordant parental allele expression (i.e., a mixture of maternally and paternally expressed informative SNPs in the same gene).…”

“…However, imprinting may have a selective advantage because it has been maintained throughout mammalian evolution [6]. The identification of the full catalog of imprinted genes in different mammalian species will greatly facilitate the understanding of the evolutionary roles of genomic imprinting [7]. …”

“…While high throughput, such studies require completion of genome sequencing and annotation, intensive bioinformatics, and careful independent validation (e.g., Sanger sequencing) to reduce false positives [7,11,18]. The recent completion of the sheep genome and improved annotation abilities provide a great opportunity to identify new imprinted genes in this understudied species.…”