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Cited by 92

The DOI matches its Crossref record; 61 of the 61 references it cites were found in a registry or an index.

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats

Schmid, Michael, Frei, Daniel, Patrignani, Andrea, Schlapbach, Ralph, Frey, Jürg E, Remus-Emsermann, Mitja N P, Ahrens, Christian H·2018·Nucleic Acids Research
DOI 10.1093/nar/gky726PMC PMC6158609

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Abstract

Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length, which contained several genes that may confer fitness advantages to the strain. Its complex genome, which also included a variable shufflon region, could not be de novo assembled with long reads produced by Pacific Biosciences' technology, but required very long reads from Oxford Nanopore Technologies. Importantly, a repeat analysis, whose results we release for over 9600 prokaryotes, indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this 'dark matter' for de novo genome assembly of prokaryotes. Several of these 'dark matter' genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assembly algorithms capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

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Crossref · 10.1093/nar/gky726

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Figures

Figure 1. Genome assembly complexity of the genus Pseudomonas. (A) Original representation of genome assembly complexity, as described by Koren and colleagues (26). The highlighted P. koreensis strain CRS05-R5 strain represents a class I genome. A total of 25 genomes are not covered in this plot, including 10 genomes with more than 300 repeats and 15 genomes whose longest repeats exceeded 30 kb (upper right box). (B) Overview of the most complex Pseudomonas strains. P. koreensis strain P19E3 is labeled (red circle) and several other strains with complex genomes, including 8 Psa strains (green).
Figure 1. Genome assembly complexity of the genus Pseudomonas. (A) Original representation of genome assembly complexity, as described by Koren and colleagues (26). The highlighted P. koreensis strain CRS05-R5 strain represents a class I genome. A total of 25 genomes are not covered in this plot, including 10 genomes with more than 300 repeats and 15 genomes whose longest repeats exceeded 30 kb (upper right box). (B) Overview of the most complex Pseudomonas strains. P. koreensis strain P19E3 is labeled (red circle) and several other strains with complex genomes, including 8 Psa strains (green).CC BY · PubMed Central
Figure 2. Length distribution of PacBio (RSII platform with BluePippin size selection, light gray) and ONT reads (MinION 1D2 libraries, dark gray). ONT can generate very long reads; we here only show 1D reads above 30 kb, as shorter reads were not used for the assembly. The percentage of reads per length bin (500 bp) is shown on the left, while the number of ONT reads longer than 90 kb is shown in the right legend. Reads longer than 150 kb (115 reads in total, longest completely mappable read 288 kb) are not shown. The three dashed lines show (from left to right) the N50 read length of PacBio subreads, the N50 read length of ONT reads above 30 kb and the length of the longest repeat in the P. koreensis P19E3 genome.
Figure 2. Length distribution of PacBio (RSII platform with BluePippin size selection, light gray) and ONT reads (MinION 1D2 libraries, dark gray). ONT can generate very long reads; we here only show 1D reads above 30 kb, as shorter reads were not used for the assembly. The percentage of reads per length bin (500 bp) is shown on the left, while the number of ONT reads longer than 90 kb is shown in the right legend. Reads longer than 150 kb (115 reads in total, longest completely mappable read 288 kb) are not shown. The three dashed lines show (from left to right) the N50 read length of PacBio subreads, the N50 read length of ONT reads above 30 kb and the length of the longest repeat in the P. koreensis P19E3 genome.CC BY · PubMed Central
Figure 3. Graph of the final high-quality P. koreensis P19E3 genome assembly. For display, the five circular elements (one chromosome, 4 plasmids) were linearized and not drawn to scale. Going from outward to inward circles: 1) ONT data assembly using Flye, 2) PacBio data assembled with Flye and 3) PacBio data assembled with HGAP3 are shown. Repeats above 30 kb are shown in the center (blue and gray bands, blue showing the longest repeat), which are also listed in Table 4; they coincide with areas where the PacBio-based assemblies were fragmented. A genomic region identified as a structural variation (ca. 5 kb) is also shown (red mark on plasmid 2). For the HGAP3 assembly with PacBio reads (innermost circle), regions are marked (in yellow) which, due to an assembly error, mapped to both chromosome and plasmid; Flye was able to resolve this region. Since plasmid 4 did not get assembled with the long read technologies, due to its short size, there is no PacBio counterpart in the PacBio tracks.
Figure 3. Graph of the final high-quality P. koreensis P19E3 genome assembly. For display, the five circular elements (one chromosome, 4 plasmids) were linearized and not drawn to scale. Going from outward to inward circles: 1) ONT data assembly using Flye, 2) PacBio data assembled with Flye and 3) PacBio data assembled with HGAP3 are shown. Repeats above 30 kb are shown in the center (blue and gray bands, blue showing the longest repeat), which are also listed in Table 4; they coincide with areas where the PacBio-based assemblies were fragmented. A genomic region identified as a structural variation (ca. 5 kb) is also shown (red mark on plasmid 2). For the HGAP3 assembly with PacBio reads (innermost circle), regions are marked (in yellow) which, due to an assembly error, mapped to both chromosome and plasmid; Flye was able to resolve this region. Since plasmid 4 did not get assembled with the long read technologies, due to its short size, there is no PacBio counterpart in the PacBio tracks.CC BY · PubMed Central
Figure 4. Overview of a shufflon region on plasmid 2. (A) The genomic region surrounding the shufflon region on plasmid 2 (top track) is shown along with gene annotations from NCBI (second track). The shufflon region is indicated as white box (third track), and the orientations of the inverted repeats are shown (as black arrows, track 4). The repeats are all pointing inwards to a position at around 102.5 kb. The coverage of mapped Illumina reads is shown (track 5). (B) Structure of the two major shufflon compositions identified. The upper version represents the sequence incorporated into the final assembly of P19E3. The lower variant exhibited extensive rearrangement of the shufflon locus (F: forward strand; R: reverse strand). (C) Consensus of inverted repeats of 19 bp length represented as a sequence logo (40).
Figure 4. Overview of a shufflon region on plasmid 2. (A) The genomic region surrounding the shufflon region on plasmid 2 (top track) is shown along with gene annotations from NCBI (second track). The shufflon region is indicated as white box (third track), and the orientations of the inverted repeats are shown (as black arrows, track 4). The repeats are all pointing inwards to a position at around 102.5 kb. The coverage of mapped Illumina reads is shown (track 5). (B) Structure of the two major shufflon compositions identified. The upper version represents the sequence incorporated into the final assembly of P19E3. The lower variant exhibited extensive rearrangement of the shufflon locus (F: forward strand; R: reverse strand). (C) Consensus of inverted repeats of 19 bp length represented as a sequence logo (40).CC BY · PubMed Central
Figure 5. Genome assembly complexity of 9331 bacteria and 293 archaea whose genomes have been completed. Blue/orange dots represent bacterial/archaeal assemblies, respectively, with the overall number of repeats as x-axis coordinate (up to 600 repeats) and the longest repeat as y-axis coordinate (up to 100 kb). The dotted lines separate the three genome assembly complexity classes; the solid black line represents the original plot dimensions by Koren and colleagues (26). The light gray zone represents genomes that potentially can be assembled with PacBio reads depending on the quality of the library and sequencing run. Above 45 kb repeat length (dark gray zone), ONT reads are expected to provide significant benefit whereas closed genomes with PacBio reads only would depend on further advances in technology. Barplots visualize the distribution of the overall number of repeats per strain on top and longest repeat per strain on the right.
Figure 5. Genome assembly complexity of 9331 bacteria and 293 archaea whose genomes have been completed. Blue/orange dots represent bacterial/archaeal assemblies, respectively, with the overall number of repeats as x-axis coordinate (up to 600 repeats) and the longest repeat as y-axis coordinate (up to 100 kb). The dotted lines separate the three genome assembly complexity classes; the solid black line represents the original plot dimensions by Koren and colleagues (26). The light gray zone represents genomes that potentially can be assembled with PacBio reads depending on the quality of the library and sequencing run. Above 45 kb repeat length (dark gray zone), ONT reads are expected to provide significant benefit whereas closed genomes with PacBio reads only would depend on further advances in technology. Barplots visualize the distribution of the overall number of repeats per strain on top and longest repeat per strain on the right.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. Motahari, 2013, Optimal DNA shotgun sequencing: Noisy reads are as good as noiseless reads, 1640
  2. Koren, 2013, Reducing assembly complexity of microbial genomes with single-molecule sequencing, Genome Biol., 14, R101, 10.1186/gb-2013-14-9-r101 · DOI

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Metadata

Authors
Schmid, M., Frei, D., Patrignani, A., Schlapbach, R., Frey, J. E., Remus-Emsermann, M. N. P., & Ahrens, C. H.
Affiliations
  • Agroscope, Molecular Diagnostics, Genomics & Bioinformatics, Wädenswil CH-8820, Switzerland
  • Functional Genomics Center Zurich, University of Zurich & ETH Zurich, Zurich CH-8057, Switzerland
  • School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand
ORCID
Ahrens, Christian H
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2018-08-23
Volume
46
Issue
17
Pages
8953-8965
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gky726
PMC
PMC6158609
Cited by
92
References
61
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Swiss National Science Foundation (31003A-156320)

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