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Reconstructing complex regions of genomes using long-read sequencing technology.

ABSTRACT: Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.

SUBMITTER: Huddleston J

PROVIDER: S-EPMC3975067 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Reconstructing complex regions of genomes using long-read sequencing technology.

Huddleston John J Ranade Swati S Malig Maika M Antonacci Francesca F Chaisson Mark M Hon Lawrence L Sudmant Peter H PH Graves Tina A TA Alkan Can C Dennis Megan Y MY Wilson Richard K RK Turner Stephen W SW Korlach Jonas J Eichler Evan E EE

Genome research 20140113 4

Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generati ...[more]

PMID: 24418700

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Project description: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.

Project description:BackgroundThe marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds.ResultsWe have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes.ConclusionDespite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is 'better' than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.

Dataset Information

Reconstructing complex regions of genomes using long-read sequencing technology.

Publications

Reconstructing complex regions of genomes using long-read sequencing technology.

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