Long-read sequencing is revolutionizing
The emergence of third‐generation sequencing (3GS; long‐reads) is bringing closer the goal of chromosome‐size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of nonmodel organisms. However, long‐read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short‐reads and long‐reads, provide an alternative efficient and cost‐effective approach to generate de novo, chromosome‐level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation are constantly being expanded and improved. This makes it difficult for nonexperts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of nonmodel organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism
- NSF-PAR ID:
- 10454698
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 20
- Issue:
- 5
- ISSN:
- 1755-098X
- Page Range / eLocation ID:
- p. 1277-1293
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract de-novo genome assemblies, with continued advancements making it more readily available for previously understudied, non-model organisms. Stony corals are one such example, with long-readde-novo genome assemblies now starting to be publicly available, opening the door for a wide array of ‘omics-based research. Here we present a newde-novo genome assembly for the endangered Caribbean star coral,Orbicella faveolata , using PacBio circular consensus reads. Our genome assembly improved the contiguity (51 versus 1,933 contigs) and complete and single copy BUSCO orthologs (93.6% versus 85.3%, database metazoa_odb10), compared to the currently available reference genome generated using short-read methodologies. Our newde-novo assembled genome also showed comparable quality metrics to other coral long-read genomes. Telomeric repeat analysis identified putative chromosomes in our scaffolded assembly, with these repeats at either one, or both ends, of scaffolded contigs. We identified 32,172 protein coding genes in our assembly through use of long-read RNA sequencing (ISO-seq) of additionalO. faveolata fragments exposed to a range of abiotic and biotic treatments, and publicly available short-read RNA-seq data. With anthropogenic influences heavily affectingO. faveolata , as well as its increasing incorporation into reef restoration activities, this updated genome resource can be used for population genomics and other ‘omics analyses to aid in the conservation of this species. -
Abstract Motivation De novo transcriptome analysis using RNA-seq offers a promising means to study gene expression in non-model organisms. Yet, the difficulty of transcriptome assembly means that the contigs provided by the assembler often represent a fractured and incomplete view of the transcriptome, complicating downstream analysis. We introduce Grouper, a new method for clustering contigs from de novo assemblies that are likely to belong to the same transcripts and genes; these groups can subsequently be analyzed more robustly. When provided with access to the genome of a related organism, Grouper can transfer annotations to the de novo assembly, further improving the clustering.
Results On de novo assemblies from four different species, we show that Grouper is able to accurately cluster a larger number of contigs than the existing state-of-the-art method. The Grouper pipeline is able to map greater than 10% more reads against the contigs, leading to accurate downstream differential expression analyses. The labeling module, in the presence of a closely related annotated genome, can efficiently transfer annotations to the contigs and use this information to further improve clustering. Overall, Grouper provides a complete and efficient pipeline for processing de novo transcriptomic assemblies.
Availability and implementation The Grouper software is freely available at https://github.com/COMBINE-lab/grouper under the 2-clause BSD license.
Supplementary information Supplementary data are available at Bioinformatics online.
-
Abstract Advances in DNA sequencing have made it feasible to gather genomic data for non‐model organisms and large sets of individuals, often using methods for sequencing subsets of the genome. Several of these methods sequence DNA associated with endonuclease restriction sites (various RAD and GBS methods). For use in taxa without a reference genome, these methods rely on
de novo assembly of fragments in the sequencing library. Many of the software options available for this application were originally developed for other assembly types and we do not know their accuracy for reduced representation libraries. To address this important knowledge gap, we simulated data from theArabidopsis thaliana andHomo sapiens genomes and comparedde novo assemblies by six software programs that are commonly used or promising for this purpose (ABySS ,CD‐HIT ,Stacks ,Stacks2 ,Velvet andVSEARCH ). We simulated different mutation rates and types of mutations, and then applied the six assemblers to the simulated data sets, varying assembly parameters. We found substantial variation in software performance across simulations and parameter settings.ABySS failed to recover any true genome fragments, andVelvet andVSEARCH performed poorly for most simulations.Stacks andStacks2 produced accurate assemblies of simulations containing SNPs, but the addition of insertion and deletion mutations decreased their performance.CD‐HIT was the only assembler that consistently recovered a high proportion of true genome fragments. Here, we demonstrate the substantial difference in the accuracy of assemblies from different software programs and the importance of comparing assemblies that result from different parameter settings. -
Abstract Background De novo phased (haplo)genome assembly using long-read DNA sequencing data has improved the detection and characterization of structural variants (SVs) in plant and animal genomes. Able to span across haplotypes, long reads allow phased, haplogenome assembly in highly outbred organisms such as forest trees. Eucalyptus tree species and interspecific hybrids are the most widely planted hardwood trees with F1 hybrids of Eucalyptus grandis and E. urophylla forming the bulk of fast-growing pulpwood plantations in subtropical regions. The extent of structural variation and its effect on interspecific hybridization is unknown in these trees. As a first step towards elucidating the extent of structural variation between the genomes of E. grandis and E. urophylla, we sequenced and assembled the haplogenomes contained in an F1 hybrid of the two species.
Findings Using Nanopore sequencing and a trio-binning approach, we assembled the separate haplogenomes (566.7 Mb and 544.5 Mb) to 98.0% BUSCO completion. High-density SNP genetic linkage maps of both parents allowed scaffolding of 88.0% of the haplogenome contigs into 11 pseudo-chromosomes (scaffold N50 of 43.8 Mb and 42.5 Mb for the E. grandis and E. urophylla haplogenomes, respectively). We identify 48,729 SVs between the two haplogenomes providing the first detailed insight into genome structural rearrangement in these species. The two haplogenomes have similar gene content, 35,572 and 33,915 functionally annotated genes, of which 34.7% are contained in genome rearrangements.
Conclusions Knowledge of SV and haplotype diversity in the two species will form the basis for understanding the genetic basis of hybrid superiority in these trees.
-
Shapiro, Beth (Ed.)Abstract In addition to including one of the most popular companion animals, species from the cat family Felidae serve as a powerful system for genetic analysis of inherited and infectious disease, as well as for the study of phenotypic evolution and speciation. Previous diploid-based genome assemblies for the domestic cat have served as the primary reference for genomic studies within the cat family. However, these versions suffered from poor resolution of complex and highly repetitive regions, with substantial amounts of unplaced sequence that is polymorphic or copy number variable. We sequenced the genome of a female F1 Bengal hybrid cat, the offspring of a domestic cat (Felis catus) x Asian leopard cat (Prionailurus bengalensis) cross, with PacBio long sequence reads and used Illumina sequence reads from the parents to phase >99.9% of the reads into the 2 species’ haplotypes. De novo assembly of the phased reads produced highly continuous haploid genome assemblies for the domestic cat and Asian leopard cat, with contig N50 statistics exceeding 83 Mb for both genomes. Whole-genome alignments reveal the Felis and Prionailurus genomes are colinear, and the cytogenetic differences between the homologous F1 and E4 chromosomes represent a case of centromere repositioning in the absence of a chromosomal inversion. Both assemblies offer significant improvements over the previous domestic cat reference genome, with a 100% increase in contiguity and the capture of the vast majority of chromosome arms in 1 or 2 large contigs. We further demonstrated that comparably accurate F1 haplotype phasing can be achieved with members of the same species when one or both parents of the trio are not available. These novel genome resources will empower studies of feline precision medicine, adaptation, and speciation.more » « less