Improving Illumina assemblies with Hi‐C and long reads: An example with the North African dromedary

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate‐pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi‐C and Dovetail Genomics Chicago libraries and long‐read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high‐quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high‐quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi‐C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome‐level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi‐C libraries increased the longest scaffold over 12‐fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50‐fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long‐read sequencing.     

Augmenting Chinese hamster genome assembly by identifying regions of high confidence

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot‐gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re‐scaffolding and gap‐filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as ”high confidence regions“ which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high‐quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines.     

Decoding the oak genome: public release of sequence data,assembly, annotation and publication strategies

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole‐genome shotgun (WGS) approach, without the use of costly and time‐consuming methods, such as fosmid or BAC clone‐based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS‐FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired‐end and mate‐pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired‐end reads and contaminants detected, resulting in a total of 17 910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.     

Orthologous comparison in a gene-rich region among grasses reveals stability in the sugarcane polyploid genome

Modern sugarcane (Saccharum spp.) is an important grass that contributes 60% of the raw sugar produced worldwide and has a high biofuel production potential. It was created about a century ago through hybridization of two highly polyploid species, namely S. officinarum and S. spontaneum. We investigated genome dynamics in this highly polyploid context by analyzing two homoeologous sequences (97 and 126 kb) in a region that has already been studied in several cereals. Our findings indicate that the two Saccharum species diverged 1.5-2 million years ago from one another and 8-9 million years ago from sorghum. The two sugarcane homoeologous haplotypes show perfect colinearity as well as high gene structure conservation. Apart from the insertion of a few retrotransposable elements, high homology was also observed for the non-transcribed regions. Relative to sorghum, the sugarcane sequences displayed colinearity, with the exception of two genes present only in sorghum, and striking homology in most non-coding parts of the genome. The gene distribution highlighted high synteny and colinearity with rice, and partial colinearity with each homoeologous maize region, which became perfect when the sequences were combined. The haplotypes observed in sugarcane may thus closely represent the ancestral Andropogoneae haplotype. This analysis of sugarcane haplotype organization at the sequence level suggests that the high ploidy in sugarcane did not induce generalized reshaping of its genome, thus challenging the idea that polyploidy quickly induces generalized rearrangement of genomes. These results also confirm the view that sorghum is the model of choice for sugarcane.     

High‐throughput genomics in sorghum: from whole‐genome resequencing to a SNP screening array

With its small, diploid and completely sequenced genome, sorghum (Sorghum bicolor L. Moench) is highly amenable to genomics‐based breeding approaches. Here, we describe the development and testing of a robust single‐nucleotide polymorphism (SNP) array platform that enables polymorphism screening for genome‐wide and trait‐linked polymorphisms in genetically diverse S. bicolor populations. Whole‐genome sequences with 6× to 12× coverage from five genetically diverse S. bicolor genotypes, including three sweet sorghums and two grain sorghums, were aligned to the sorghum reference genome. From over 1 million high‐quality SNPs, we selected 2124 Infinium Type II SNPs that were informative in all six source genomes, gave an optimal Assay Design Tool (ADT) score, had allele frequencies of 50% in the six genotypes and were evenly spaced throughout the S. bicolor genome. Furthermore, by phenotype‐based pool sequencing, we selected an additional 876 SNPs with a phenotypic association to early‐stage chilling tolerance, a key trait for European sorghum breeding. The 3000 attempted bead types were used to populate half of a dual‐species Illumina iSelect SNP array. The array was tested using 564 Sorghum spp. genotypes, including offspring from four unrelated recombinant inbred line (RIL) and F₂ populations and a genetic diversity collection. A high call rate of over 80% enabled validation of 2620 robust and polymorphic sorghum SNPs, underlining the efficiency of the array development scheme for whole‐genome SNP selection and screening, with diverse applications including genetic mapping, genome‐wide association studies and genomic selection.     

Sorghum genome sequencing by methylation filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.     

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole‐genome shotgun sequencing of the nuclear genome of flax. Seven paired‐end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep‐coverage (approximately 94× raw, approximately 69× filtered) short‐sequence reads (44–100 bp), produced a set of scaffolds with N₅₀ = 694 kb, including contigs with N₅₀ = 20.1 kb. The contig assembly contained 302 Mb of non‐redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole‐genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis‐assembly of regions at the genome scale. A total of 43 384 protein‐coding genes were predicted in the whole‐genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K_s) observed within duplicate gene pairs was consistent with a recent (5–9 MYA) whole‐genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam‐A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole‐genome shotgun short‐sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.     

Exploiting rice–sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 × E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice–sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.     

Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.     

Draft genome of an iconic Red Sea reef fish,the blacktail butterflyfish (Chaetodon austriacus): current status and its characteristics

Butterflyfish are among the most iconic of the coral reef fishes and represent a model system to study general questions of biogeography, evolution and population genetics. We assembled and annotated the genome sequence of the blacktail butterflyfish (Chaetodon austriacus), an Arabian region endemic species that is reliant on coral reefs for food and shelter. Using available bony fish (superclass Osteichthyes) genomes as a reference, a total of 28 926 high‐quality protein‐coding genes were predicted from 13 967 assembled scaffolds. The quality and completeness of the draft genome of C. austriacus suggest that it has the potential to serve as a resource for studies on the co‐evolution of reef fish adaptations to the unique Red Sea environment, as well as a comparison of gene sequences between closely related congeneric species of butterflyfish distributed more broadly across the tropical Indo‐Pacific.     

A Second Generation Integrated Map of the Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Genome: Analysis of Conserved Synteny with Model Fish Genomes

DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries were generated to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is composed of 167,989 clones of which 158,670 are assembled into contigs and 9,319 are singletons. The number of contigs was reduced from 4,173 to 3,220. End sequencing of clones from the new libraries generated a total of 11,958 high quality sequence reads. The end sequences were used to develop 238 new microsatellites of which 42 were added to the genetic map. Conserved synteny between the rainbow trout genome and model fish genomes was analyzed using 188,443 BAC end sequence (BES) reads. The fractions of BES reads with significant BLASTN hits against the zebrafish, medaka, and stickleback genomes were 8.8%, 9.7%, and 10.5%, respectively, while the fractions of significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 6.2%, 5.8%, and 5.5%, respectively. The overall number of unique regions of conserved synteny identified through grouping of the rainbow trout BES into fingerprinting contigs was 2,259, 2,229, and 2,203 for stickleback, medaka, and zebrafish, respectively. These numbers are approximately three to five times greater than those we have previously identified using BAC paired ends. Clustering of the conserved synteny analysis results by linkage groups as derived from the integrated physical and genetic map revealed that despite the low sequence homology, large blocks of macrosynteny are conserved between chromosome arms of rainbow trout and the model fish species.     

Castor bean organelle genome sequencing and worldwide genetic diversity analysis

Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade.     

A genome-wide BAC end-sequence survey of sugarcane elucidates genome composition, and identifies BACs covering much of the euchromatin

BAC-end sequences (BESs) of hybrid sugarcane cultivar R570 are presented. A total of 66,990 informative BESs were obtained from 43,874 BAC clones. Similarity search using a variety of public databases revealed that 13.5 and 42.8 % of BESs match known gene-coding and repeat regions, respectively. That 11.7 % of BESs are still unmatched to any nucleotide sequences in the current public databases despite the fact that a close relative, sorghum, is fully sequenced, indicates that there may be many sugarcane-specific or lineage-specific sequences. We found 1,742 simple sequence repeat motifs in 1,585 BESs, spanning 27,383 bp in length. As simple sequence repeat markers derived from BESs have some advantages over randomly generated markers, these may be particularly useful for comparing BAC-based physical maps with genetic maps. BES and overgo hybridization information was used for anchoring sugarcane BAC clones to the sorghum genome sequence. While sorghum and sugarcane have extensive similarity in terms of genomic structure, only 2,789 BACs (6.4 %) could be confidently anchored to the sorghum genome at the stringent threshold of having both-end information (BESs or overgos) within 300 Kb. This relatively low rate of anchoring may have been caused in part by small- or large-scale genomic rearrangements in the Saccharum genus after two rounds of whole genome duplication since its divergence from the sorghum lineage about 7.8 million years ago. Limiting consideration to only low-copy matches, 1,245 BACs were placed to 1,503 locations, covering ~198 Mb of the sorghum genome or about 78 % of the estimated 252 Mb of euchromatin. BESs and their analyses presented here may provide an early profile of the sugarcane genome as well as a basis for BAC-by-BAC sequencing of much of the basic gene set of sugarcane.     

Sequencing chromosome 5D of Aegilops tauschii and comparison with its allopolyploid descendant bread wheat (Triticum aestivum)

Flow cytometric sorting of individual chromosomes and chromosome‐based sequencing reduces the complexity of large, repetitive Triticeae genomes. We flow‐sorted chromosome 5D of Aegilops tauschii, the D genome donor of bread wheat and sequenced it by Roche 454 GS FLX platform to approximately 2.2x coverage. Repetitive sequences represent 81.09% of the survey sequences of this chromosome, and Class I retroelements are the prominent type, with a particular abundance of LTR/Gypsy superfamily. Nonrepetitive sequences were assembled to cover 17.76% of the total chromosome regions. Up to 6188 nonrepetitive gene loci were predicted to be encoded by the 5D chromosome. The numbers and chromosomal distribution patterns of tRNA genes suggest abundance in tRNA^Lys and tRNA^Met species, while the nonrepetitive assembly reveals tRNA^Ala species as the most abundant type. A comparative analysis of the genomic sequences of bread wheat and Aegilops chromosome 5D indicates conservation of gene content. Orthologous unique genes, matching Aegilops 5D sequences, numbered 3730 in barley, 5063 in Brachypodium, 4872 in sorghum and 4209 in rice. In this study, we provide a chromosome‐specific view into the structure and organization of the 5D chromosome of Ae. tauschii, the D genome ancestor of bread wheat. This study contributes to our understanding of the chromosome‐level evolution of the wheat genome and presents a valuable resource in wheat genomics due to the recent hybridization of Ae. tauschii genome with its tetraploid ancestor.     

Assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley

Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third‐generation sequencer (PacBio), we de novo assembled a 4.84‐Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59‐Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high‐confidence’ genes, 31 564 were validated by RNA‐sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low‐diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.     

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor? using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 10⁸ cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 10⁸ cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 10⁸ cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10⁸ and 10⁸ cells/mL was performed using cells from a perfusion run at 10⁸ cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013