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

Nature Protocols volume 7, pages 1260–1284 (2012)Cite this article

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Abstract

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.

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Figure 1
Figure 2: The 182 scaffolds in the E. coli assembly contain 342 gaps after being mapped to the reference genome.
Figure 3
Figure 4: The basic workflow of the protocol is shown for two common use-cases: for de novo assembly and when a reference genome is available.
Figure 5: Output of the PAGIT test script displayed in ACT.
Figure 6: Example of models in the E. coli example that were not transferred in RATT displayed in ACT.
Figure 7: View of an example of a frameshift in a gene model, visualized using ACT.

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Acknowledgements

We thank L. Chappel for testing and checking the protocol; T. Carver for helping with the installation of the Virtual Machine; and M. Hunt for testing the virtual machine. T.D.O. was supported by the European Union 7th framework European Virtual Institute of Malaria Research (EVIMalaR); I.J.T., S.A.A. and M.B. were supported by the Wellcome Trust (grant number: 098051).

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Authors and Affiliations

  1. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK

    Martin T Swain, Isheng J Tsai, Samual A Assefa, Chris Newbold, Matthew Berriman & Thomas D Otto

  2. Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais Campus, Aberystwyth, UK

    Martin T Swain

  3. Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK

    Chris Newbold

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  1. Martin T Swain
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Contributions

M.T.S., T.D.O., C.N. and M.B. conceived and executed the examples. T.D.O., M.T.S., I.J.T. and S.A.A. conceived and wrote the installation procedures. All authors were involved with the writing of the manuscript.

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Correspondence to Thomas D Otto.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

PAGIT: two worked examples. Here we give a synopsis of the work-flow used for the two examples discussed in the "Anticipated Results" section. (DOCX 31 kb)

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Swain, M., Tsai, I., Assefa, S. et al. A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs. Nat Protoc 7, 1260–1284 (2012). https://doi.org/10.1038/nprot.2012.068

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