Abstract
Motivation
Rapid development in long-read sequencing and scaffolding technologies is accelerating the production of reference-quality assemblies for large eukaryotic genomes. However, ...haplotype divergence in regions of high heterozygosity often results in assemblers creating two copies rather than one copy of a region, leading to breaks in contiguity and compromising downstream steps such as gene annotation. Several tools have been developed to resolve this problem. However, they either focus only on removing contained duplicate regions, also known as haplotigs, or fail to use all the relevant information and hence make errors.
Results
Here we present a novel tool, purge_dups, that uses sequence similarity and read depth to automatically identify and remove both haplotigs and heterozygous overlaps. In comparison with current tools, we demonstrate that purge_dups can reduce heterozygous duplication and increase assembly continuity while maintaining completeness of the primary assembly. Moreover, purge_dups is fully automatic and can easily be integrated into assembly pipelines.
Availability and implementation
The source code is written in C and is available at https://github.com/dfguan/purge_dups.
Supplementary information
Supplementary data are available at Bioinformatics online.
The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects ...the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.
Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution
. Here we generate and analyse ...reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.
Abstract
Background
False duplications in genome assemblies lead to false biological conclusions. We quantified false duplications in popularly used previous genome assemblies for platypus, zebra ...finch, and Anna’s Hummingbird, and their new counterparts of the same species generated by the Vertebrate Genomes Project, of which the Vertebrate Genomes Project pipeline attempted to eliminate false duplications through haplotype phasing and purging. These assemblies are among the first generated by the Vertebrate Genomes Project where there was a prior chromosomal level reference assembly to compare with.
Results
Whole genome alignments revealed that 4 to 16% of the sequences are falsely duplicated in the previous assemblies, impacting hundreds to thousands of genes. These lead to overestimated gene family expansions. The main source of the false duplications is heterotype duplications, where the haplotype sequences were relatively more divergent than other parts of the genome leading the assembly algorithms to classify them as separate genes or genomic regions. A minor source is sequencing errors. Ancient ATP nucleotide binding gene families have a higher prevalence of false duplications compared to other gene families. Although present in a smaller proportion, we observe false duplications remaining in the Vertebrate Genomes Project assemblies that can be identified and purged.
Conclusions
This study highlights the need for more advanced assembly methods that better separate haplotypes and sequence errors, and the need for cautious analyses on gene gains.
Multicellular eukaryotes have evolved a range of mechanisms for immune recognition. A widespread family involved in innate immunity are the NACHT-domain and leucine-rich-repeat-containing (NLR) ...proteins. Mammals have small numbers of NLR proteins, whereas in some species, mostly those without adaptive immune systems, NLRs have expanded into very large families. We describe a family of nearly 400 NLR proteins encoded in the zebrafish genome. The proteins share a defining overall structure, which arose in fishes after a fusion of the core NLR domains with a B30.2 domain, but can be subdivided into four groups based on their NACHT domains. Gene conversion acting differentially on the NACHT and B30.2 domains has shaped the family and created the groups. Evidence of positive selection in the B30.2 domain indicates that this domain rather than the leucine-rich repeats acts as the pathogen recognition module. In an unusual chromosomal organization, the majority of the genes are located on one chromosome arm, interspersed with other large multigene families, including a new family encoding zinc-finger proteins. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system.
...we have developed a system to track individual regions that are under review. The primary assembly unit contains sequences for the non-redundant haploid assembly; this includes the scaffolds that ...make up the chromosome sequence as well as unplaced and unlocalized scaffolds that are thought to represent novel sequence (not shown in this picture).\n Additionally, we wish to engage the research and clinical communities to identify regions that require targeted effort and to incorporate information from groups performing detailed work on specific loci.
Many short-read genome assemblies have been found to be incomplete and contain mis-assemblies. The Vertebrate Genomes Project has been producing new reference genome assemblies with an emphasis on ...being as complete and error-free as possible, which requires utilizing long reads, long-range scaffolding data, new assembly algorithms, and manual curation. A more thorough evaluation of the recent references relative to prior assemblies can provide a detailed overview of the types and magnitude of improvements.
Here we evaluate new vertebrate genome references relative to the previous assemblies for the same species and, in two cases, the same individuals, including a mammal (platypus), two birds (zebra finch, Anna's hummingbird), and a fish (climbing perch). We find that up to 11% of genomic sequence is entirely missing in the previous assemblies. In the Vertebrate Genomes Project zebra finch assembly, we identify eight new GC- and repeat-rich micro-chromosomes with high gene density. The impact of missing sequences is biased towards GC-rich 5'-proximal promoters and 5' exon regions of protein-coding genes and long non-coding RNAs. Between 26 and 60% of genes include structural or sequence errors that could lead to misunderstanding of their function when using the previous genome assemblies.
Our findings reveal novel regulatory landscapes and protein coding sequences that have been greatly underestimated in previous assemblies and are now present in the Vertebrate Genomes Project reference genomes.
The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken ...genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.
Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic ...fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.
Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics ...depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.
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