The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.

The Pacific oyster (Crassostrea gigas) is a bivalve mollusc with vital roles in coastal ecosystems and aquaculture globally. While extensive genomic tools are available for C. gigas, highly contiguous reference genomes are required to support both fundamental and applied research. Herein we report the creation and annotation of a chromosome-level assembly for C. gigas. High-coverage long- and short-read sequence data generated on Pacific Biosciences and Illumina platforms were used to generate an initial assembly, which was then scaffolded into 10 pseudo-chromosomes using both Hi-C sequencing and a high-density linkage map. The assembly has a scaffold N50 of 58.4 Mb and a contig N50 of 1.8 Mb, representing a step advance on the previously published C. gigas assembly. Annotation based on Pacific Biosciences Iso-Seq and Illumina RNA-Seq resulted in identification of ∼30,000 putative protein-coding genes. Annotation of putative repeat elements highlighted an enrichment of Helitron rolling-circle transposable elements, suggesting their potential role in shaping the evolution of the C. gigas genome. This new chromosome-level assembly will be an enabling resource for genetics and genomics studies to support fundamental insight into bivalve biology, as well as for selective breeding of C. gigas in aquaculture.

Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5′-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences. We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5′ cap selection was performed on the embryo library to provide methodological comparison. From these Iso-Seq sequencing projects, we have identified 60 k transcripts and 29 k genes within the chicken transcriptome. Of these, more than 20 k are novel lncRNA transcripts with ~3 k classified as sense exonic overlapping lncRNA, which is a class that is underrepresented in many vertebrate annotations. The relative proportion of alternative transcription events revealed striking similarities between the chicken and human transcriptomes while also providing explanations for previously observed genomic differences. Our results indicate that the chicken transcriptome is similar in complexity compared to human, and provide insights into other vertebrate biology. Our methodology demonstrates the potential of Iso-Seq sequencing to rapidly expand our knowledge of transcriptomics.

The contribution of regulatory versus protein change to adaptive evolution has long been controversial. In principle, the rate and strength of adaptation within functional genetic elements can be quantified on the basis of an excess of nucleotide substitutions between species compared to the neutral expectation or from effects of recent substitutions on nucleotide diversity at linked sites. Here, we infer the nature of selective forces acting in proteins, their UTRs and conserved noncoding elements (CNEs) using genome-wide patterns of diversity in wild house mice and divergence to related species. By applying an extension of the McDonald-Kreitman test, we infer that adaptive substitutions are widespread in protein-coding genes, UTRs and CNEs, and we estimate that there are at least four times as many adaptive substitutions in CNEs and UTRs as in proteins. We observe pronounced reductions in mean diversity around nonsynonymous sites (whether or not they have experienced a recent substitution). This can be explained by selection on multiple, linked CNEs and exons. We also observe substantial dips in mean diversity (after controlling for divergence) around protein-coding exons and CNEs, which can also be explained by the combined effects of many linked exons and CNEs. A model of background selection (BGS) can adequately explain the reduction in mean diversity observed around CNEs. However, BGS fails to explain the wide reductions in mean diversity surrounding exons (encompassing ∼100 Kb, on average), implying that there is a substantial role for adaptation within exons or closely linked sites. The wide dips in diversity around exons, which are hard to explain by BGS, suggest that the fitness effects of adaptive amino acid substitutions could be substantially larger than substitutions in CNEs. We conclude that although there appear to be many more adaptive noncoding changes, substitutions in proteins may dominate phenotypic evolution. We present an analysis of the genome sequences of multiple wild house mice. Wild house mice are about ten times more genetically diverse than humans, reflecting the large effective population size of the species. This manifests itself as more effective natural selection acting against deleterious mutations and favouring advantageous mutations in mice than in humans. We show that there are strong signals of adaptive evolution at many sites in the genome. We estimate that 80% of adaptive changes in the genome are in gene regulatory elements and only 20% are in protein-coding genes. We find that nucleotide diversity is markedly reduced close to gene regulatory elements and protein-coding gene sequences. The reductions around regulatory elements can be explained by selection purging deleterious mutations that occur in the elements themselves, but this process only partially explains the diversity reductions around protein-coding genes. Recurrent adaptive evolution, which can also cause local reductions in diversity via selective sweeps, may be necessary to fully explain the patterns in diversity that we observe surrounding genes. Although most adaptive molecular evolution appears to be regulatory, adaptive phenotypic change may principally be driven by structural change in proteins.

Protein-coding sequences make up only about 1% of the mammalian genome. Much of the remaining 99% has been long assumed to be junk DNA, with little or no functional significance. Here, we show that in hominids, a group with historically low effective population sizes, all classes of noncoding DNA evolve more slowly than ancestral transposable elements and so appear to be subject to significant evolutionary constraints. Under the nearly neutral theory, we expected to see lower levels of selective constraints on most sequence types in hominids than murids, a group that is thought to have a higher effective population size. We found that this is the case for many sequence types examined, the most extreme example being 5′UTRs, for which constraint in hominids is only about one-third that of murids. Surprisingly, however, we observed higher constraints for some sequence types in hominids, notably 4-fold sites, where constraint is more than twice as high as in murids. This implies that more than about one-fifth of mutations at 4-fold sites are effectively selected against in hominids. The higher constraint at 4-fold sites in hominids suggests a more complex protein-coding gene structure than murids and indicates that methods for detecting selection on protein-coding sequences (e.g., using the d_N/d_S ratio), with 4-fold sites as a neutral standard, may lead to biased estimates, particularly in hominids. Our constraint estimates imply that 5.4% of nucleotide sites in the human genome are subject to effective negative selection and that there are three times as many constrained sites within noncoding sequences as within protein-coding sequences. Including coding and noncoding sites, we estimate that the genomic deleterious mutation rate U = 4.2. The mutational load predicted under a multiplicative model is therefore about 99% in hominids.