Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

BACKGROUND: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. METHODOLOGY/PRINCIPAL FINDINGS: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. CONCLUSION: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.

Recent progress in understanding the genomic architecture of sexual conflict.

Genomic conflict between the sexes over shared traits is widely assumed to be resolved through the evolution of sex-biased expression and the subsequent emergence of sexually dimorphic phenotypes. However, while there is support for a broad relationship between genome-wide patterns of expression level and sexual conflict, recent studies suggest that sex differences in the nature and strength of interactions between loci are instead key to conflict resolution. Furthermore, the advent of new technologies for measuring and perturbing expression means we now have much more power to detect genomic signatures of sexual conflict. Here, we review our current understanding of the genomic architecture of sexual conflict in the light of these new studies and highlight the potential for novel approaches to address outstanding knowledge gaps.

Detecting signatures of selection on gene expression.

A substantial amount of phenotypic diversity results from changes in gene expression levels and patterns. Understanding how the transcriptome evolves is therefore a key priority in identifying mechanisms of adaptive change. However, in contrast to powerful models of sequence evolution, we lack a consensus model of gene expression evolution. Furthermore, recent work has shown that many of the comparative approaches used to study gene expression are subject to biases that can lead to false signatures of selection. Here we first outline the main approaches for describing expression evolution and their inherent biases. Next, we bridge the gap between the fields of phylogenetic comparative methods and transcriptomics to reinforce the main pitfalls of inferring selection on expression patterns and use simulation studies to show that shifts in tissue composition can heavily bias inferences of selection. We close by highlighting the multi-dimensional nature of transcriptional variation and identifying major unanswered questions in disentangling how selection acts on the transcriptome.