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Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens

Identifieur interne : 000336 ( Istex/Corpus ); précédent : 000335; suivant : 000337

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens

Auteurs : Bastian Greshake ; Simonida Zehr ; Francesco Dal Grande ; Anjuli Meiser ; Imke Schmitt ; Ingo Ebersberger

Source :

RBID : ISTEX:387D63BC2B6BA93BF2A016EA284C49570D55B60A

Abstract

Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all‐purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.

Url:
DOI: 10.1111/1755-0998.12463

Links to Exploration step

ISTEX:387D63BC2B6BA93BF2A016EA284C49570D55B60A

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<p>Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers
<hi rend="italic">Omega</hi>
and
<hi rend="italic">MetaVelvet</hi>
, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast,
<hi rend="italic">
<hi rend="fc">MIRA</hi>
</hi>
, an all‐purpose overlap assembler, and
<hi rend="fc">SPA</hi>
des, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen
<hi rend="italic">Lasallia pustulata</hi>
demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont
<hi rend="italic">Trebouxia sp</hi>
. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.</p>
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<b>Fig. S1. </b>
<fc>NG</fc>
50 vs.
<fc>NGA</fc>
50 for the assemblies generated by the six assemblers across all 11 data sets.</caption>
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<b>Fig. S2.</b>
Coverage distributions for the contigs of the fungal (A) and algal (B) fraction in the
<i>Lasallia pustulata</i>
metagenome assembly.</caption>
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<p>
<b>Fig. S3. </b>
<i>k mer</i>
coverage plot of the 0:10 twin set for k = 141.</p>
<p>
<b>Fig. S4. </b>
<i>k mer</i>
coverage plot of the 1:9 twin set for k = 131.</p>
<p>
<b>Fig. S5. </b>
<i>k mer</i>
coverage plot of the 2:8 twin set for k = 131.</p>
<p>
<b>Fig. S6. </b>
<i>k mer</i>
coverage plot of the 3:7 twin set for k = 141.</p>
<p>
<b>Fig. S7. </b>
<i>k mer</i>
coverage plot of the 4:6 twin set for k = 141.</p>
<p>
<b>Fig. S8. </b>
<i>k mer</i>
coverage plot of the 5:5 twin set for k = 91.</p>
<p>
<b>Fig. S9. </b>
<i>k mer</i>
coverage plot of the 6:4 twin set for k = 131.</p>
<p>
<b>Fig. S10. </b>
<i>k mer</i>
coverage plot of the 7:3 twin set for k = 51.</p>
<p>
<b>Fig. S11. </b>
<i>k mer</i>
coverage plot of the 8:2 twin set for k = 51.</p>
<p>
<b>Fig. S12. </b>
<i>k mer</i>
coverage plot of the 9:1 twin set for k = 151.</p>
<p>
<b>Fig. S13. </b>
<i>k mer</i>
coverage plot of the 10:0 twin set for k = 131.</p>
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<p>
<b>Table S1.</b>
Quast report of the 0:10 set.</p>
<p>
<b>Table S2.</b>
Quast report of the 1:9 set.</p>
<p>
<b>Table S3.</b>
Quast report of the 2:8 set.</p>
<p>
<b>Table S4.</b>
Quast report of the 3:7 set.</p>
<p>
<b>Table S5.</b>
Quast report of the 4:6 set.</p>
<p>
<b>Table S6.</b>
Quast report of the 5:5 set.</p>
<p>
<b>Table S7.</b>
Quast report of the 6:4 set.</p>
<p>
<b>Table S8.</b>
Quast report of the 7:3 set.</p>
<p>
<b>Table S9.</b>
Quast report of the 8:2 set.</p>
<p>
<b>Table S10.</b>
Quast report of the 9:1 set.</p>
<p>
<b>Table S11.</b>
Quast report of the 10:0 set.</p>
<p>
<b>Table S12.</b>
Quast report of the
<i>Lasallia pustulata</i>
metagenomic data set.</p>
<p>
<b>Table S13.</b>
Quast report of the fungal contigs of the
<i>Lasallia pustulata</i>
data set.</p>
<p>
<b>Table S14.</b>
Quast report of the algal contigs of the
<i>Lasallia pustulata</i>
data set.</p>
<p>
<b>Table S15.</b>
Quast report of the bacterial contigs of the
<i>Lasallia pustulata</i>
data set.</p>
<p>
<b>Table S16.</b>
Quast report of the 10× coverage algal data.</p>
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<p>Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers
<i>Omega</i>
and
<i>MetaVelvet</i>
, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast,
<i>
<fc>MIRA</fc>
</i>
, an all‐purpose overlap assembler, and
<fc>SPA</fc>
des, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen
<i>Lasallia pustulata</i>
demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont
<i>Trebouxia sp</i>
. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.</p>
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<abstract>Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all‐purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.</abstract>
<note type="additional physical form">Fig. S1. NG50 vs. NGA50 for the assemblies generated by the six assemblers across all 11 data sets.Fig. S2. Coverage distributions for the contigs of the fungal (A) and algal (B) fraction in the Lasallia pustulata metagenome assembly.Fig. S3. k mer coverage plot of the 0:10 twin set for k = 141. Fig. S4. k mer coverage plot of the 1:9 twin set for k = 131. Fig. S5. k mer coverage plot of the 2:8 twin set for k = 131. Fig. S6. k mer coverage plot of the 3:7 twin set for k = 141. Fig. S7. k mer coverage plot of the 4:6 twin set for k = 141. Fig. S8. k mer coverage plot of the 5:5 twin set for k = 91. Fig. S9. k mer coverage plot of the 6:4 twin set for k = 131. Fig. S10. k mer coverage plot of the 7:3 twin set for k = 51. Fig. S11. k mer coverage plot of the 8:2 twin set for k = 51. Fig. S12. k mer coverage plot of the 9:1 twin set for k = 151. Fig. S13. k mer coverage plot of the 10:0 twin set for k = 131.Table S1. Quast report of the 0:10 set. Table S2. Quast report of the 1:9 set. Table S3. Quast report of the 2:8 set. Table S4. Quast report of the 3:7 set. Table S5. Quast report of the 4:6 set. Table S6. Quast report of the 5:5 set. Table S7. Quast report of the 6:4 set. Table S8. Quast report of the 7:3 set. Table S9. Quast report of the 8:2 set. Table S10. Quast report of the 9:1 set. Table S11. Quast report of the 10:0 set. Table S12. Quast report of the Lasallia pustulata metagenomic data set. Table S13. Quast report of the fungal contigs of the Lasallia pustulata data set. Table S14. Quast report of the algal contigs of the Lasallia pustulata data set. Table S15. Quast report of the bacterial contigs of the Lasallia pustulata data set. Table S16. Quast report of the 10× coverage algal data.Appendix S1. Supplementary text: General concepts of sequence assembly based on overlap layout and de Bruijn graphs, respectively.</note>
<note type="funding">Landes‐Offensive zur Entwicklung Wissenschaftlich‐Ökonomischer Exzellenz (LOEWE) of Hesse's Ministry of Higher Education, Research</note>
<note type="funding">EU - No. PITM‐GA‐2013‐607607; </note>
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<title>Ahmadjian V (1993) The Lichen Symbiosis. John Wiley and Sons Inc, New York, NY.</title>
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