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EVE 161 Winter 2018 Class 17
This document summarizes a study that reconstructed 7,903 bacterial and archaeal genomes from over 1,500 public metagenomes. Key findings include: - The genomes increase phylogenetic diversity of bacterial and archaeal trees by over 30% and provide first representatives for 17 bacterial and 3 archaeal candidate phyla. - 245 genomes were recovered from the Patescibacteria superphylum. - The genomes vary substantially in quality, with 43.5% considered near-complete, 43.8% medium quality, and 12.7% partial. - The genomes expand representation of underrepresented phyla like Aminicenantes, Gemmatimonadetes, and Lentisphaera
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EVE 161 Winter 2018 Class 17
- 1. EVE 161: Microbial Phylogenomics CaseStudy 1000s of MAGs UC Davis, Winter 2018 Instructor: Jonathan Eisen Teaching Assistant: Cassie Ettinger !1
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- 4. ARTICLES DOI: 10.1038/s41564-017-0012-7 S equencing ofmicrobial genomes has accelerated with reduc- tions in sequencing costs, and public repositories now contain nearly 70,000 bacterial and archaeal genomes. The majority phyla previously lacking genomic representatives27–29 , including the Patescibacteria superphylum4 , which has subsequently been referred to as the ‘Candidate Phyla Radiation’ (CPR) as it may con- Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life Donovan H. Parks , Christian Rinke , Maria Chuvochina, Pierre-Alain Chaumeil, Ben J. Woodcroft, Paul N. Evans, Philip Hugenholtz * and Gene W. Tyson* Challenges in cultivating microorganisms have limited the phylogenetic diversity of currently available microbial genomes. This is being addressed by advances in sequencing throughput and computational techniques that allow for the cultivation-inde- pendent recovery of genomes from metagenomes. Here, we report the reconstruction of 7,903 bacterial and archaeal genomes from >1,500 public metagenomes. All genomes are estimated to be ≥50% complete and nearly half are ≥90% complete with ≤5% contamination. These genomes increase the phylogenetic diversity of bacterial and archaeal genome trees by >30% and provide the first representatives of 17 bacterial and three archaeal candidate phyla. We also recovered 245 genomes from the Patescibacteria superphylum (also known as the Candidate Phyla Radiation) and find that the relative diversity of this group varies substantially with different protein marker sets. The scale and quality of this data set demonstrate that recovering genomes from metagenomes provides an expedient path forward to exploring microbial dark matter. Corrected: Author correction
- 5. ARTICLES NATURE MICROBIOLOGY formthe UBA data set as they met our filtering criteria of having an estimated quality ≥50 (defined as the estimated completeness of a Taxonomic distribution of UBA genomes. The phylogenetic rela- tionships of the UBA genomes were determined across bacterial 50 60 70 80 90 100 Completeness (%) 0 2 4 6 8 10Contamination(%) Near complete (3,438 genomes; 43.5%) Medium quality (3,459 genomes; 43.8%) Partial (1,006 genomes; 12.7%) 12% 33% a 05101520 Standard tRNA count 0 5 10 15 20 Genomes(%) 0 100 200 300 400 500 No. of scaffolds 0 4 8 12 Genomes(%) 83% of genomes have ≤200 scaffolds b c 75% of genomes have ≥15 standard tRNAs Fig. 1 | Assessment of genome quality. a, Estimated completeness and contamination of 7,903 genomes recovered from public metagenomes. Genome quality was defined as completeness− 5×contamination, and only genomes with a quality of ≥50 were retained. Near-complete genomes (completeness ≥90%; contamination ≤5%) are shown in red, medium-quality genomes (completeness ≥70%; contamination ≤10%) in blue, and partial genomes (completeness ≥50%; contamination ≤4%) in grey. Histograms along the x and y axes show the percentage of genomes at varying levels of completeness and contamination, respectively. Notably, only 171 of the 7,903 (2.2%) UBA genomes have >5% contamination. b, Number of scaffolds comprising each of the 7,903 genomes with colours indicating genome quality. c, Number of tRNAs for each of the 20 standard amino acids identified within each of the 7,903 genomes with colours indicating genome quality.
- 6. ARTICLESNATURE MICROBIOLOGY 38 classes(18.0%) within this domain. Similarily, the archaeal UBA Only UBP9, UAP2 and UAP3 could be further taxonomically Proteobacteria (2,101) Deltaproteo. (1-3) (226) Dadabacteria (2) MBNT15 (2) UBP10 (2) Nitrospirae_1 (19) Chrysio. (1) NC10 Rokubacteria Modulibacteria Nitrospirae (23) Nitrospinae (5) Acidobacteria (35) Aminicenantes (10) Chlorobi Bacteroidetes (1,513) Ignavibacteriae (29) Calditrichaeota (5) Marinimicrobia (46), UBP11 (1) Fibrobacteres (17), Gemmatimonadetes (39) Zixibacteria, UBP14 (1), TA06, UBP1 (3), UBP2 (6) Cloacimonetes (31), Hyd24-12 (5), WOR-3 (10) Lentisphaerae (12), Chlamydiae, UBP17 (1) Verrucomicrobia (178) Planctomycetes (61) Hydrogenedentes (2) Poribacteria Elusimicrobia (23) Aerophobetes, UBP5 (2) Omnitrophica (14), UBP3 (4), UBP4 (2) Firmicutes (1,666) Armatimonadetes (24) UBP13 (1) Actinobacteria (336) Deinococcus-thermus (6) Chloroflexi (182) UBP9/SHA-109 (8) UBP12 (1) Cyanobacteria (48)Thermotogae (11) Acetothermia (10) Dictyoglomi Coprothermo. Caldiserica (5) Synergistetes (47) Atribacteria (5) UBP16 (1) UBP15 (1) Epsilonproteobacteria (52) Calescamantes Spirochaetes (135) UBP8 (5) UBP7 (2) Patescibacteria (245) 'Candidate phyla radiation' UBP6 (4) Aquificae (1) Deferribacteres (5) Fusobacteria (8) Dependentiae (1) Latescibacteria (4) 0.1 FCB PVC Terrabacteria Fig. 2 | Distribution of UBA genomes across 76 bacterial phyla. The maximum likelihood tree was inferred from the concatenation of 120 proteins and spans a dereplicated set of 5,273 UBA and 14,304 NCBI genomes. Phyla containing UBA genomes are shown in green with the number of UBA genomes indicated in parentheses. Candidate phyla consisting only of UBA genomes are shown in red and have been named Uncultured Bacterial Phylum 1 to 17 (UBP1–UBP17). The Tenericutes and Thermodesulfobacteria lineages are not shown as they branch within the Firmicutes and Deltaproteobacteria, respectively. The Patescibacteria/CPR is shown as a single lineage for clarity and to illustrate its similarity to well-characterized phyla. The Nitrospirae and Deltaproteobacteria were found to be polyphyletic and an underscore and numerical identifier is used to distinguish between lineages. The Deltaproteobacteria are polyphyletic due to the placement of the Dadabacteria. All named lineages have bootstrap support of ≥75%, with the exception of the Chrysiogenetes (55%), Firmicutes (1%) and Verrucomicrobia (52%). The complete tree with support values is available in Newick format (Supplementary File 1). Fig. 2 | Distribution of uBA genomes across 76 bacterial phyla. The maximum likelihood tree was inferred from the concatenation of 120 proteins and spans a dereplicated set of 5,273 UBA and 14,304 NCBI genomes. Phyla containing UBA genomes are shown in green with the number of UBA genomes indicated in parentheses. Candidate phyla consisting only of UBA genomes are shown in red and have been named Uncultured Bacterial Phylum 1 to 17 (UBP1–UBP17). The Tenericutes and Thermodesulfobacteria lineages are not shown as they branch within the Firmicutes and Deltaproteobacteria, respectively. The Patescibacteria/CPR is shown as a single lineage for clarity and to illustrate its similarity to well-characterized phyla. The Nitrospirae and Deltaproteobacteria were found to be polyphyletic and an underscore and numerical identifier is used to distinguish between lineages. The Deltaproteobacteria are polyphyletic due to the placement of the Dadabacteria. All named lineages have bootstrap support of ≥75%, with the exception of the Chrysiogenetes (55%), Firmicutes (1%) and Verrucomicrobia (52%). The complete tree with support values is available in Newick format (Supplementary File 1).
- 7. TICLES NATURE MICROBIOLOGY Euryarchaeota(538) Crenarchaeota (11) Korarchaeota Verstraetearchaeota (2) Thaumarchaeota (26) Aigarchaeota Bathyarchaeota (3) Thorarchaeota Lokiarchaeota UAP3/AAG (1) Woesearchaeota_2 (2) Pacearchaeota (3) Woesearchaeota (6) UAP1 (2) Parvarchaeota (11) Nanoarchaeota Nanohaloarchaeota Aenigmarchaeota Micrarchaeota (12) Diapherotrites (1) UAP2/MHVG(4) 0.1 DPANN TACK
- 8. including 245 UBAgenomes) and Firmicutes (25,992 genomes, including 1,666 UBA genomes) is 11.2% and 25.1%, respectively (Fig. 5). Restricting results to bacterial UBA genomes meeting our near-complete or medium-quality criteria still results in PGs of ~17% and 30%, respectively. The near-complete and medium- quality archaeal UBA genomes provide a phylogenetic gain of 10% and 29%, respectively (Supplementary Table 8). Genomic representation of several bacterial lineages was greatly expanded by the UBA genomes (Fig. 5). The bacterial phyla with the largest increase in PD were the underrepresented Aminicenantes, Gemmatimonadetes, Lentisphaerae and Omnitrophica lineages (PG of >75%). Over 75% of the bacterial UBA genomes belong to the Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. These as the Micrarchaeota, Pacearchaeota and Woesearchaeota which all had a PG of >35%. Improved genomic representatives within several lineages. There are 12 bacterial phyla where the UBA genomes are estimated to be the highest-quality representatives (Supplementary Table 17). Among these is the Aminicenantes, where the number of avail- able genomes increased from 37 to 47 with the addition of the UBA genomes, and the highest-quality genome improved from 86.9% to 91.9% complete, with the five highest-quality genomes all being UBA genomes. There are currently seven Hydrogenedentes genomes (five NCBI and two UBA), with the two highest-quality representatives being UBA genomes and appreciably improving UAP3/AAG (1) Woesearchaeota_2 (2) Pacearchaeota (3) Woesearchaeota (6) UAP1 (2) Parvarchaeota (11) Nanoarchaeota Nanohaloarchaeota Aenigmarchaeota Micrarchaeota (12) Diapherotrites (1) DPANN Fig. 3 | Distribution of UBA genomes across 21 archaeal phyla. The maximum likelihood tree was inferred from the concatenation of 122 proteins and spans a dereplicated set of 453 UBA and 630 NCBI genomes. Phyla containing UBA genomes are shown in green with the number of UBA genomes indicated in parentheses. Candidate phyla consisting only of UBA genomes are shown in red and have been named Uncultured Archaeal Phylum 1 to 3 (UAP1–UAP3). The phylum Woesearchaeota was found to be polyphyletic and an underscore and numerical identifier is used to distinguish between lineages. All named lineages have bootstrap support ≥90%, with the exception of the Crenarchaeota (73%). The complete tree with support values is available in Newick format (Supplementary File 2). MHVG, Marine Hydrothermal Vent Group; AAG, Ancient Archaeal Group.
- 9. ARICROBIOLOGY a b5,107 100 80 60 40 20 0 100 529 287 181 116 77 54 51 99 161 328 39 22 16 4 4 80 60 40 20 0 0.0 0.2 Lineages(%) Lineages(%) 0.40.6 0.8 1.0 0.0 0.2 UBA NCBI UBA and NCBI 0.4 0.6 0.8 1.01.2 Phylum Class Order Family Genus Phylum Class Order Family Genus 1,927 1,735 4,150 821 867 357 379 151 76 76 36 14 3 Number of bacterial lineages Number of archaeal lineages Mean branch length (substitutions per site) Mean branch length (substitutions per site) e of lineages of equal evolutionary distance represented exclusively by UBA genomes. a, Percentage of bacterial lineag UBA genomes, genomes at NCBI, or both data sets as defined on the basis of mean branch length to extant taxa. Mean s calculated for all internal nodes and lineages defined at a specific threshold to establish lineages of equal evolutionary cates the threshold used to define lineages (0 = extant taxa, far right = root of tree), the top x axis indicates the number anch length thresholds, and the left y axis indicates the percentage of lineages. The right y axis indicates different taxono percentiles of the distribution of mean branch length values for these ranks shown by black lines. The mean of each dist long with the number of lineages at this value. b, Analogous plot to a, but for archaeal lineages. The phylum distribution ota is the only archaeal phylum with phylum-level resolution (that is, containing multiple classes). Plots were determine rees inferred from the concatenation of 120 bacterial and 122 archaeal proteins, respectively. ARTICLESNATURE MICROBIOLOGY representative within the Micrarchaeota (Micrarchaeum acidiphi- lum ARMAN-2) from 84% complete to 91% complete, while add- the phylogenetic diversity of bacterial and archaeal genome trees by >30% through the addition of 7,280 bacterial and 623 archaeal a b5,107 100 80 60 40 20 0 100 529 287 181 116 77 54 51 99 161 328 39 22 16 4 4 80 60 40 20 0 0.0 0.2 Lineages(%) Lineages(%) 0.4 0.6 0.8 1.0 0.0 0.2 UBA NCBI UBA and NCBI 0.4 0.6 0.8 1.01.2 Phylum Class Order Family Genus Phylum Class Order Family Genus 1,927 1,735 4,150 821 867 357 379 151 76 76 36 14 3 Number of bacterial lineages Number of archaeal lineages Mean branch length (substitutions per site) Mean branch length (substitutions per site) Fig. 4 | Percentage of lineages of equal evolutionary distance represented exclusively by UBA genomes. a, Percentage of bacterial lineages represented exclusively by the UBA genomes, genomes at NCBI, or both data sets as defined on the basis of mean branch length to extant taxa. Mean branch length to extant taxa was calculated for all internal nodes and lineages defined at a specific threshold to establish lineages of equal evolutionary distance. The bottom x axis indicates the threshold used to define lineages (0 = extant taxa, far right = root of tree), the top x axis indicates the number of lineages at different mean branch length thresholds, and the left y axis indicates the percentage of lineages. The right y axis indicates different taxonomic ranks, with the 5th and 90th percentiles of the distribution of mean branch length values for these ranks shown by black lines. The mean of each distribution is shown as a black circle along with the number of lineages at this value. b, Analogous plot to a, but for archaeal lineages. The phylum distribution is a single point, as the Euryarchaeota is the only archaeal phylum with phylum-level resolution (that is, containing multiple classes). Plots were determined across the domain-specific trees inferred from the concatenation of 120 bacterial and 122 archaeal proteins, respectively.
- 10. ARTICLES NATURE MI NCBIUBA NCBI UBA Total PD Taxon PD UBA PG NCBI PD UBA PD UBA PG/PD Total Dereplicated Relative Archaea 100.0% 30.7% UAP (all) 1.1% 100.0% Euryarchaeota 52.7% 34.8% Marine Group II 9.2% 85.9% Micrarchaeota 2.4% 73.4% Pacearchaeota 2.7% 37.9% Thaumarchaeota 6.4% 40.8% Woesearchaeota 4.4% 42.9% Bacteria 100.0% 33.0% UBP (all) 1.1% 1.1% Acetothermia 0.1% 71.8% Aminicenantes 0.2% 81.0% Armatimonadetes 0.4% 67.2% Bacteroidetes 9.5% 47.4% Caldiserica 0.2% 74.3% Chloroflexi 2.7% 73.1% Dadabacteria 0.1% 74.8% Elusimicrobia 0.3% 70.5% Firmicutes 25.1% 32.2% Gemmatimonadetes 0.2% 78.6% Hyd24-12 0.1% 61.4% Latescibacteria 0.1% 64.7% Lentisphaerae 0.3% 75.8% Omnitrophica 0.3% 89.3% Patescibacteria / CPR 11.2% 25.8% Proteobacteria 20.5% 25.0% Verrucomicrobia 825 623 630 453 0 7 0 7 534 538 422 369 12 206 12 206 1 12 1 12 5 3 5 3 49 26 44 26 7 6 7 6 67,034 7,244 14,304 5,273 0 46 0 46 1 10 1 6 4 10 4 10 12 24 6 13 1,324 1,513 941 1,057 1 5 1 4 61 182 50 169 1 2 1 2 3 23 3 14 24,326 1,666 3,433 1,296 4 39 3 37 3 5 3 5 1 4 1 3 2 12 2 12 1 14 1 14 811 245 811 245 28,860 2,101 4,889 1,272 45 178 44 166 1.9% 65.5% Fig. 5 | Phylogenetic diversity and gain for select bacterial and archaeal phyla. The 7,903 UBA genomes cover considerable phylogenetic diversity (PD) and contribute substantial phylogenetic gain (PG) relative to genomes from RefSeq/GenBank release 76. PD and PG were assessed on the domain-specific trees inferred from the concatenation of 120 bacterial and 122 archaeal proteins. The bar charts give (1) the total PD for each lineage relative to the PD of the entire domain, (2) the PD of the NCBI and UBA genomes relative to the PD of the lineage, and (3) the PG contributed by the UBA genomes within the lineage. The 17 UBP and 3 UAP lineages were collapsed together to show their combined phylogenetic diversity and gain. Marine Group II, a lineage within the Euryarchaeota, has been included because it comprises the majority of euryarchaeotal UBA genomes.
- 11. ARTINATURE MICROBIOLOGY c rps16d rps23 f bac120e rps16 a bac120 b bac120 3,270 100 80 60 40 20 0 100 80 60 40 20 0 0.0 Lineages(%) Lineages(%) 0.2 0.4 0.6 Mean branch length (substitutions per site) Mean branch length (substitutions per site) 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1,282 583 279 Number of lineages Number of lineages 133 68 68 291 616 1,162 2,646 34 12 2 5,107 1,927 821 867 1,735 4,150 357 379 151 76 76 36 14 3 Phylum Class Order Family Genus Other bacteriaUBA exclusive CPR UBA exclusive CPR 100 80 60 40 20 0 0.0 Lineages(%) 0.2 0.4 0.6 Mean branch length (substitutions per site) 0.8 1.0 3,454 1,197 476 Number of lineages 179 67 71 475 924 2,031 4,187 27 5 Phylum Class Order Family Genus 100 80 60 40 20 0 0.0 Lineages(%) 0.2 0.4 0.6 Mean branch length (substitutions per site) 0.8 1.0 3,209 1,064 411 Number of lineages 160 67 69 449 845 1,872 4,422 22 3 Phylum Class Order Family Genus 100 80 60 40 20 0 0.0 Lineages(%) 0.2 0.4 0.6 Mean branch length (substitutions per site) 0.8 1.0 3,454 1,197 476 Number of lineages 179 67 71 475 924 2,031 4,187 27 5 Phylum Class Order Family Genus Other bacteria Bacteroidetes Proteobacteria Actinobacteria Firmicutes CPR 100 80 60 40 20 0 0.0 Lineages(%) 0.2 0.4 0.6 Mean branch length (substitutions per site) 0.8 1.0 1.2 5,107 1,927 357 821 Number of lineages 151 76 76 379 867 1,735 4,150 14 36 3 Phylum Class Order Family Genus Phylum Class Order Family Genus Fig. 6 | Percentage of lineages of equal evolutionary distance within the CPR. a, Percentage of CPR lineages within the bac120 tree without the Fig. 6 | Percentage of lineages of equal evolutionary distance within the CPR. a, Percentage of CPR lineages within the bac120 tree without the addition of the UBA genomes. The maximum percentage of phylum-level lineages within the CPR occurs at a mean branch length of 0.85, as denoted by the dashed grey line. The remaining figure elements are the same as in Fig. 4. b, Analogous plot to a, with the addition of the UBA genomes (maximum = 0.85). c,d, Percentage of CPR lineages within the rp1 and rp2 trees, respectively, with the maximum percentage of phylum-level lineages within the CPR denoted with a dashed grey line (rp1 = 0.69; rp2 = 0.76) e,f, Percentage of CPR, Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes lineages within the rp1 and bac120 trees, respectively. The trees span 14,290 (a), 19,198 (b and f), 18,081 (c and e) and 18,226 (d) genomes.
- 12. Recently, the diversityof the CPR was explored in the context of a genome tree inferred from 16 ribosomal proteins where it was divided into 36 named phyla and shown to represent approximately 50% of bacterial lineages of equal phylum-level evolutionary distance30. Our analyses using a 120 concatenated proteins contrast with this view, as the CPR is shown to comprise ~25% of phylum- level lineages under the same criterion (Fig. 6b and Supplementary Fig. 7). This suggests that ribosomal proteins within CPR organisms may be evolving atypically relative to other proteins, perhaps as a result of their unusual ribosome composition and the presence of self-splicing introns and proteins being encoded within their rRNA genes10 . These contrasting views of the diversity of the CPR are equally valid and probably reflect the unique biology of the organ- isms within this group.