Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar ‘Fielder’

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar ‘Fielder’, an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.     

De novo Assembly of highly diverse viral populations

ABSTRACT: BACKGROUND: Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and, as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. RESULTS: We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/viral-genomics-analysis-software CONCLUSIONS: We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.     

Long-read sequence assembly: a technical evaluation in barley

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

A greatly improved reference genome sequence of barley was assembled from accurate long reads.     

An improved genome reference for the African cichlid,Metriaclima zebra

Background

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.

Results

Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.

Conclusions

Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1930-5) contains supplementary material, which is available to authorized users.     

Chromosome-scale haplotype-phased genome assemblies of the male and female lines of wild asparagus (Asparagus kiusianus), a dioecious plant species

Asparagus kiusianus is a disease-resistant dioecious plant species and a wild relative of garden asparagus (Asparagus officinalis). To enhance A. kiusianus genomic resources, advance plant science, and facilitate asparagus breeding, we determined the genome sequences of the male and female lines of A. kiusianus. Genome sequence reads obtained with a linked-read technology were assembled into four haplotype-phased contig sequences (∼1.6 Gb each) for the male and female lines. The contig sequences were aligned onto the chromosome sequences of garden asparagus to construct pseudomolecule sequences. Approximately 55,000 potential protein-encoding genes were predicted in each genome assembly, and ∼70% of the genome sequence was annotated as repetitive. Comparative analysis of the genomes of the two species revealed structural and sequence variants between the two species as well as between the male and female lines of each species. Genes with high sequence similarity with the male-specific sex determinant gene in A. officinalis, MSE1/AoMYB35/AspTDF1, were presented in the genomes of the male line but absent from the female genome assemblies. Overall, the genome sequence assemblies, gene sequences, and structural and sequence variants determined in this study will reveal the genetic mechanisms underlying sexual differentiation in plants, and will accelerate disease-resistance breeding in garden asparagus.     

Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene‐rich regions. Gene‐enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene‐enrichment strategy, we have compared assemblies using methyl‐filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA‐seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single‐nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.     

An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies

Background

Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture.

Results

The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene.

Conclusion

High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1103) contains supplementary material, which is available to authorized users.     

Single molecule sequencing-guided scaffolding and correction of draft assemblies

Background

Although single molecule sequencing is still improving, the lengths of the generated sequences are inevitably an advantage in genome assembly. Prior work that utilizes long reads to conduct genome assembly has mostly focused on correcting sequencing errors and improving contiguity of de novo assemblies.

Results

We propose a disassembling-reassembling approach for both correcting structural errors in the draft assembly and scaffolding a target assembly based on error-corrected single molecule sequences. To achieve this goal, we formulate a maximum alternating path cover problem. We prove that this problem is NP-hard, and solve it by a 2-approximation algorithm.

Conclusions

Our experimental results show that our approach can improve the structural correctness of target assemblies in the cost of some contiguity, even with smaller amounts of long reads. In addition, our reassembling process can also serve as a competitive scaffolder relative to well-established assembly benchmarks.

     

Polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads to improve genomic analyses

Oxford Nanopore sequencing has been widely used to achieve complete genomes of bacterial pathogens. However, the error rates of Oxford Nanopore long reads are high. Various polishing algorithms using Illumina short reads to correct the errors in Oxford Nanopore long-read assemblies have been developed. The impact of polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads on improving genomic analyses was evaluated using both simulated and real reads. Ten species (10 strains) were selected for simulated reads, while real reads were tested on 11 species (11 strains). Oxford Nanopore long reads were assembled with Unicycler to produce a draft assembly, followed by three rounds of polishing with Illumina short reads using two polishing tools, Pilon and NextPolish. One round of NextPolish polishing generated genome completeness and accuracy parameters similar to the reference genomes, whereas two or three rounds of Pilon polishing were needed, though contiguity remained unchanged after polishing. The polished assemblies of Escherichia coli O157:H7, Salmonella Typhimurium, and Cronobacter sakazakii with simulated reads did not provide accurate plasmid identifications. One round of NextPolish polishing was needed for accurately identifying plasmids in Staphylococcus aureus and E. coli O26:H11 with real reads, whereas one and two rounds of Pilon polishing were necessary for these two strains, respectively. Polishing failed to provide an accurate antimicrobial resistance (AMR) genotype for S. aureus with real reads. One round of polishing recovered an accurate AMR genotype for Klebsiella pneumoniae with real reads. The reference genome and draft assembly of Citrobacter braakii with real reads differed, which carried blaCMY-83 and fosA6, respectively, while both genes were present after one round of polishing. However, polishing did not improve the assembly of E. coli O26:H11 with real reads to achieve numbers of virulence genes similar to the reference genome. The draft and polished assemblies showed a phylogenetic tree topology comparable with the reference genomes. For multilocus sequence typing and pan-genome analyses, one round of NextPolish polishing was sufficient to obtain accurate results, while two or three rounds of Pilon polishing were needed. Overall, NextPolish outperformed Pilon for polishing the Oxford Nanopore long-read assemblies of bacterial pathogens, though both polishing strategies improved genomic analyses compared to the draft assemblies.     

Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.     

AIMIE: a web-based environment for detection and interpretation of significant sequence motifs in prokaryotic genomes

Motivation: Genomes contain biologically significant informationthat extends beyond that encoded in genes. Some of this informationrelates to various short dispersed repeats distributed throughoutthe genome. The goal of this work was to combine tools for detectionof statistically significant dispersed repeats in DNA sequenceswith tools to aid development of hypotheses regarding theirpossible physiological functions in an easy-to-use web-basedenvironment. Results: Ab Initio Motif Identification Environment (AIMIE)was designed to facilitate investigations of dispersed sequencemotifs in prokaryotic genomes. We used AIMIE to analyze theEscherichia coli and Haemophilus influenzae genomes in orderto demonstrate the utility of the new environment. AIMIE detectedrepeated extragenic palindrome (REP) elements, CRISPR repeats,uptake signal sequences, intergenic dyad sequences and severalother over-represented sequence motifs. Distributional patternsof these motifs were analyzed using the tools included in AIMIE. Availability: AIMIE and the related software can be accessedat our web site http://www.cmbl.uga.edu/software.html. Contact: mrazek{at}uga.edu Associate Editor: Alex Bateman     

Harnessing cross-species alignment to discover SNPs and generate a draft genome sequence of a bighorn sheep (Ovis canadensis)

Background

Whole genome sequences (WGS) have proliferated as sequencing technology continues to improve and costs decline. While many WGS of model or domestic organisms have been produced, a growing number of non-model species are also being sequenced. In the absence of a reference, construction of a genome sequence necessitates de novo assembly which may be beyond the ability of many labs due to the large volumes of raw sequence data and extensive bioinformatics required. In contrast, the presence of a reference WGS allows for alignment which is more tractable than assembly. Recent work has highlighted that the reference need not come from the same species, potentially enabling a wide array of species WGS to be constructed using cross-species alignment. Here we report on the creation a draft WGS from a single bighorn sheep (Ovis canadensis) using alignment to the closely related domestic sheep (Ovis aries).

Results

Two sequencing libraries on SOLiD platforms yielded over 865 million reads, and combined alignment to the domestic sheep reference resulted in a nearly complete sequence (95% coverage of the reference) at an average of 12x read depth (104 SD). From this we discovered over 15 million variants and annotated them relative to the domestic sheep reference. We then conducted an enrichment analysis of those SNPs showing fixed differences between the reference and sequenced individual and found significant differences in a number of gene ontology (GO) terms, including those associated with reproduction, muscle properties, and bone deposition.

Conclusion

Our results demonstrate that cross-species alignment enables the creation of novel WGS for non-model organisms. The bighorn sheep WGS will provide a resource for future resequencing studies or comparative genomics.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1618-x) contains supplementary material, which is available to authorized users.     

Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools

Background

Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality.

Results

In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS.

Conclusions

By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-439) contains supplementary material, which is available to authorized users.     

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent validation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

ConFunc--functional annotation in the twilight zone

Motivation: The success of genome sequencing has resulted inmany protein sequences without functional annotation. We presentConFunc, an automated Gene Ontology (GO)-based protein functionprediction approach, which uses conserved residues to generatesequence profiles to infer function. ConFunc split sets of sequencesidentified by PSI-BLAST into sub-alignments according to theirGO annotations. Conserved residues are identified for each GOterm sub-alignment for which a position specific scoring matrixis generated. This combination of steps produces a set of feature(GO annotation) derived profiles from which protein functionis predicted. Results: We assess the ability of ConFunc, BLAST and PSI-BLASTto predict protein function in the twilight zone of sequencesimilarity. ConFunc significantly outperforms BLAST & PSI-BLASTobtaining levels of recall and precision that are not obtainedby either method and maximum precision 24% greater than BLAST.Further for a large test set of sequences with homologues oflow sequence identity, at high levels of presicision, ConFuncobtains recall six times greater than BLAST. These results demonstratethe potential for ConFunc to form part of an automated genomicsannotation pipeline. Availability: http://www.sbg.bio.ic.ac.uk/confunc Contact: m.sternberg{at}imperial.ac.uk Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Dmitrij Frishman