Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read Sequencing Technology

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.     

The Fast Changing Landscape of Sequencing Technologies and Their Impact on Microbial Genome Assemblies and Annotation

Background

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation.

Methodology/Principal Findings

In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis.

Conclusion

These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).     

Reducing assembly complexity of microbial genomes with single-molecule sequencing

Background

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.

Results

To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.

Conclusions

Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.     

Whole Genome Complete Resequencing of Bacillus subtilis Natto by Combining Long Reads with High-Quality Short Reads

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.     

A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs

Genome projects now produce draft assemblies within weeks owing to advanced high-throughput sequencing technologies. For milestone projects such as Escherichia coli or Homo sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects, and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT) that is used to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence and exploit reference genomes (if available) in order to improve scaffolding and generating annotations. The protocol is most accessible for bacterial and small eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes ～24 h: it doubles the average contig size and annotates over 4,300 gene models.     

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.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Sealer: a scalable gap-closing application for finishing draft genomes

Background

While next-generation sequencing technologies have made sequencing genomes faster and more affordable, deciphering the complete genome sequence of an organism remains a significant bioinformatics challenge, especially for large genomes. Low sequence coverage, repetitive elements and short read length make de novo genome assembly difficult, often resulting in sequence and/or fragment “gaps” – uncharacterized nucleotide (N) stretches of unknown or estimated lengths. Some of these gaps can be closed by re-processing latent information in the raw reads. Even though there are several tools for closing gaps, they do not easily scale up to processing billion base pair genomes.

Results

Here we describe Sealer, a tool designed to close gaps within assembly scaffolds by navigating de Bruijn graphs represented by space-efficient Bloom filter data structures. We demonstrate how it scales to successfully close 50.8 % and 13.8 % of gaps in human (3 Gbp) and white spruce (20 Gbp) draft assemblies in under 30 and 27 h, respectively – a feat that is not possible with other leading tools with the breadth of data used in our study.

Conclusion

Sealer is an automated finishing application that uses the succinct Bloom filter representation of a de Bruijn graph to close gaps in draft assemblies, including that of very large genomes. We expect Sealer to have broad utility for finishing genomes across the tree of life, from bacterial genomes to large plant genomes and beyond. Sealer is available for download at https://github.com/bcgsc/abyss/tree/sealer-release.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0663-4) contains supplementary material, which is available to authorized users.     

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N₅₀ of 10,384 bp and scaffold N₅₀ of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.     

Sequencing intractable DNA to close microbial genomes

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.     

How incorrect annotations evolve--the case of short ORFs

The draft of the human genome sequence is still incomplete. The outstanding tasks include filling in some gaps, finalizing the assembly of short sequences, improving sequence accuracy and correctly identifying coding regions. However, a closely related problem that receives little attention is the substantial number of incorrect annotations that have penetrated some of the widely used databases. This article illustrates this problem using the example of ubiquitin genes, and draws some conclusions that apply to false annotations in other short open reading frames (ORFs). Although the focus is on the human genome, other genomes are equally prone to similar propagation of false annotations.     

Comparative Analysis of Begonia Plastid Genomes and Their Utility for Species-Level Phylogenetics

Recent, rapid radiations make species-level phylogenetics difficult to resolve. We used a multiplexed, high-throughput sequencing approach to identify informative genomic regions to resolve phylogenetic relationships at low taxonomic levels in Begonia from a survey of sixteen species. A long-range PCR method was used to generate draft plastid genomes to provide a strong phylogenetic backbone, identify fast evolving regions and provide informative molecular markers for species-level phylogenetic studies in Begonia.     

Improving draft assemblies by iterative mapping and assembly of short reads to eliminate gaps

Advances in sequencing technology allow genomes to be sequenced at vastly decreased costs. However, the assembled data frequently are highly fragmented with many gaps. We present a practical approach that uses Illumina sequences to improve draft genome assemblies by aligning sequences against contig ends and performing local assemblies to produce gap-spanning contigs. The continuity of a draft genome can thus be substantially improved, often without the need to generate new data.     

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Genome size and microsatellites: the effect of nuclear size on amplification potential.

Although the frequency of microsatellite DNA regions generally increases with increasing genome size, genome size has a negative effect on polymerase chain reaction (PCR) amplification. Thus, researchers developing sets of PCR primers, as is commonly done for microsatellite DNA regions, may encounter greater difficulty when working with species that have larger genomes. I investigated the effect of genome size on overall amplification success using data from nine different metazoan taxa. The proportion of primer sets that did not amplify PCR products was strongly and positively correlated with the haploid C value of the target species. Increasing genome size may affect amplification success negatively because of a decrease in target:nontarget DNA or by dilution of the available primer pool by nonspecific binding.     

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.     

Draft versus finished sequence data for DNA and protein diagnostic signature development

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10⁻³–10⁻⁵ (~8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ~1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.     

TUCAN/CARDINAL and DRAL participate in a common pathway for modulation of NF-kappaB activation

Prior to genome sequencing, information on base composition (GC level) and its variation in mammalian genomes could be obtained using density gradient ultracentrifugation. Analyses using this approach led to the conclusion that mammalian genomes are organized into mosaics of fairly homogeneous regions, called isochores. We present an initial compositional overview of the chromosomes of the recently available draft human genome sequence, in the form of color-coded moving window plots and corresponding GC level histograms. Results obtained from the draft human genome sequence agree well with those obtained or deduced earlier from CsCl experiments. The draft sequence now permits the visualization of the mosaic organization of the human genome at the DNA sequence level.