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

By A. English, S. Richards, Yi Han, Min Wang, V. Vee, Jiaxin Qu, X. Qin, D. Muzny, Jeffrey G. Reid, K. Worley and 1 more. PLoS ONE, 2012

896 citations recorded by the source corpus.

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.

Subfields: Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/d32d7aa802a0c199724560f41ede127059fb899d

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Citations in the corpus

Next generation sequencing technology: Advances and applications. (2014, methodology): “have used PacBio data for upgrading existing draft genome assemblies derived from Illumina sequencing data by looking for reads that extend into or cover gaps in the assembly [56].”
A chromosome-level genome assembly for the Pacific oyster Crassostrea gigas (2020, neutral): “Gaps were closed with PBJelly (PBJelly, RRID:SCR 012091) [54] and again error corrected using the short-read Illumina data using Pilon [44].”
The real cost of sequencing: scaling computation to keep pace with data generation (2016, background): “However, higher sequencing error rates for longer reads require novel assembly algorithms [35 – 38].”
Finding the Genomic Basis of Local Adaptation: Pitfalls, Practical Solutions, and Future Directions (2016, neutral): “In addition, reference genomes based on short-read technologies can be greatly improved by including some data from longer-read platforms to generate a hybrid genome assembly (English et al. 2012; Koren et al. 2012).”
Pathways to de‐extinction: how close can we get to resurrection of an extinct species? (2017, background): “…DNA sequences are very short, technologies that either sequence long DNA molecules [e.g. Pacific Biosciences long-read sequencing technology (English et al. 2012)] or that require long molecules to prepare DNA sequencing libraries for short-read sequencing platforms (e.g. ‘Chicago’…”
SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information (2014, methodology): “Finally dedicated tools have been developed to close gaps within scaffolds using PacBio reads, among which PBJelly [17].”
Culture-independent molecular tools for soil and rhizosphere microbiology (2013, methodology): “[179] presented new software (PBJelly) for upgrading genome assemblies based on long-read sequence form Pacific Bioscience RS.”
Piercing the dark matter: bioinformatics of long-range sequencing and mapping (2018, methodology): “A final approach is to use low-coverage long reads for scaffolding or filling gaps within an existing assembly using tools such as PBJelly or Cobbler 58,59 .”
De novo assembly of haplotype-resolved genomes with trio binning (2018, positive): “For example, the partitioned read sets can be reused for haplotype-specific gap filling 45 and consensus polishing 46, 47 , and we have shown that polishing with haplotype-specific reads achieves a more accurate consensus sequence.”
Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology (2018, background): “Another system, developed by Oxford Nanopore and called nanopore sequencing technologies, is able to generate approximately 100-kb reads (356).”
Chloroplast genomes: diversity, evolution, and applications in genetic engineering (2016, methodology): “The low accuracy (~85 % of the raw data) of the long reads produced by the PacBio platform [45] can be corrected by combining the latest chemistry with a hierarchical genome assembly process algorithm; accuracy rates as high as 99.”