Measurement(s)DNA • genome • sequence_assembly • sequence feature annotationTechnology Type(s)DNA sequencing • sequence assembly process • sequence annotationFactor Type(s)strainSample Characteristic - OrganismStreptomyces
Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11791323
Streptomyces are Gram-positive bacteria of significant industrial importance due to their ability to produce a wide range of antibiotics and bioactive secondary metabolites. Recent advances in genome mining have revealed that Streptomyces genomes possess a large number of unexplored silent secondary metabolite biosynthetic gene clusters (smBGCs). This indicates that Streptomyces genomes continue to be an invaluable source for new drug discovery. Here, we present high-quality genome sequences of 22 Streptomyces species and eight different Streptomyces venezuelae strains assembled by a hybrid strategy exploiting both long-read and short-read genome sequencing methods. The assembled genomes have more than 97.4% gene space completeness and total lengths ranging from 6.7 to 10.1 Mbp. Their annotation identified 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs on average. In silico prediction of smBGCs identified a total of 922 clusters, including many clusters whose products are unknown. We anticipate that the availability of these genomes will accelerate discovery of novel secondary metabolites from Streptomyces and elucidate complex smBGC regulation.
Background & SummaryWith the rapid emergence of antibiotic microbial resistance (AMR) to all major classes of antibiotics and the decline in number of potential candidates for new antibiotics, there is a pressing need for the discovery of novel antibacterial compounds1. Streptomyces, soil dwelling gram-positive bacteria, continue to be promising microorganisms for the production of clinically important secondary metabolites, including not only antibiotics, but also antiviral, antifungal, and antiparasitic agents, and antitumorals and immunosuppressant compounds2. Streptomyces are distinguished by their complex life cycle and high G + C content (often over 70%) in their linear genomes3,4. Traditionally, drug discovery from Streptomyces has been based on bioactivity screening followed by mass spectrometry and NMR-based molecular identification5. However, recent advances in genomics-based approaches revealed that most of the secondary metabolite biosynthetic gene clusters (smBGCs) of streptomycetes are inactive under laboratory conditions, suggesting that the ability of streptomycetes to produce secondary metabolites has been under-estimated5,6. Each Streptomyces species has the genetic potential to produce more than 30 secondary metabolites on average, which are diverse and differ between species7,8. Considering Streptomyces is the largest genus of actinobacteria with approximately 900 species characterized so far, streptomycetes are a valuable resource for the discovery of novel secondary metabolites9.SmBGCs, especially polyketide and non-ribosomal peptide synthetase types, are often composed of extraordinarily long genes (>5 kb) encoding multi-modular enzymes with repetitive domain structures. Therefore, accurate gene annotations based on high quality genome sequences are essential for the precise identification of smBGCs10. Gene annotation with the high quality genome of S. clavuligerus revealed that 30% out of a total of 7,163 protein coding genes were incorrectly annotated in the previous draft genome of S. clavuligerus containing ambiguous and inaccurate nucleotides, indicating the importance of high quality genome sequences11. In addition, high quality genome sequences are essential for multi-omics analysis, which facilitates the understanding of the complex regulation on smBGCs and rational engineering for increasing secondary metabolites production11,12.Among the 1,614 streptomycetes genomes that have been deposited in the NCBI Assembly database to date (as of 9th December 2019), only 189 and 35 assemblies were designated as complete genome level and chromosome level, respectively. More than 86% of assemblies were draft-quality genome sequences, which contain fragmented multiple contigs or ambiguous sequences4,13–15. One of the main obstacles to obtaining high quality genomic information of streptomycetes is the low fidelity of sequencing techniques when dealing with high G w C genomes and frequently repetitive sequences such as terminal inverted repeats13. In addition, since streptomycetes have linear chromosome, it is difficult to confirm the completeness of the assembled chromosome.In this study, we present the high-quality genome sequences of 30 streptomycetes, increasing the total number of reported complete Streptomyces genome by about 10%. The target streptomycetes were 22 Streptomyces type strains and eight different Streptomyces venezuelae strains, most of which are currently used as industrial strains for producing various bioactive compounds. We applied hybrid assembly strategy with long-read (PacBio) and short-read (Illumina) sequencing techniques to obtain complete genome sequences. PacBio sequencing provides long reads of several kb in length which allows the readthrough of regions with low complexity, enabling the assembly of repetitive regions, which are difficult to assemble by using Illumina sequencing reads, even with the high coverage data16. However, Illumina sequencing provides reads with a lower error rate compared to the PacBio sequencing, and assembled contigs based on the Illumina sequencing reads are not simply a subset of the contigs from PacBio sequencing reads13,17. Therefore, reconciling PacBio and Illumina sequencing methods enables one to generate more complete genomes by overcoming the shortcomings of each method. During the genome assembly using reads from PacBio (0.46~5.18 Gbp) and Illumina (0.5~3.0 Gbp) sequencing, we constructed 6.7 to 10.1 Mbp of streptomycetes genomes, most of which consist of single chromosomes with 72% G + C contents on average. Inaccurate sequences in the assembled genome were corrected using Illumina sequencing reads. The complete streptomycetes genomes have more than 97.4% gene space completeness and on average 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs were annotated. Finally, based on the complete genome sequences and annotations, we predicted a total of 922 smBGCs. The complete genome sequences and newly determined smBGCs in this study should prove to be a fundamental resource for understanding the genetic basis of streptomycetes and for discovering novel secondary metabolites.MethodsGenomic DNA (gDNA) extractionTotal 30 streptomycetes were purchased from Korean Collection for Type Cultures (KCTC, Korea). A stock of streptomycetes were inoculated to 50 mL of liquid culture medium with 0.16 g mL−1 of glass beads (3 ± 0.3 mm diameter) in 250 mL baffled flask and grown at 30 °C in a 200 rpm orbital shaker. Each streptomycetes was grown in one of four different culture medium, R5(–) medium (25 mM TES (pH 7.2), 103 g L−1 sucrose, 1% (w/v) glucose, 5 g L−1 yeast extract, 10.12 g L−1 MgCl2∙6H2O, 0.25 g L−1 K2SO4, 0.1 g L−1 casamino acids, 0.08 g L−1 ZnCl2, 0.4 mg L−1 FeCl3, 0.02 mg L−1 CuCl2∙2H2O, 0.02 mg L−1 MnCl2∙4H2O, 0.02 mg L−1 Na2B4O7∙10H2O, and 0.02 mg L−1 (NH4)6Mo7O24∙4H2O), 1 × sporulation medium (3.33 g L−1 glucose, 1 g L−1 yeast extract, 1 g L−1 beef extract, 2 g L−1 tryptose, and 0.006 g L−1 FeSO4∙7H2O), YEME medium (340 g L−1 sucrose, 10 g L−1 glucose, 3 g L−1 yeast extract, 5 g L−1 bacto peptone, and 3 g L−1 oxoid malt extract), and MYM medium (4 g L−1 maltose, 4 g L−1 yeast extract, 10 g L−1 malt extract). For gDNA extraction, 25 mL cultured cells were harvested at the exponential growth phase and washed twice with same volume of 10 mM EDTA, followed by the lysozyme (10 mg mL−1) treatment at 37 °C for 45 min. gDNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Quality and quantity of extracted gDNA samples were evaluated using 1% agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), respectively.Short-read (Illumina) genome sequencingFor construction of short-read genome sequencing library, 2.5 μg of gDNA was sheared to approximately 350 bp by a Covaris instrument (Covaris Inc., Woburn, MA, USA) with the following conditions; Power 175, Duty factor 20%, C. burst 200, Time 23 s, 8 times. The library was constructed using a TruSeq DNA PCR-Free LT kit (Illumina Inc., San Diego, CA, USA) following manufacturer’s instruction. Briefly, the fragmented DNA samples were cleaned and end-repaired, followed by the adaptor ligation and bead-based size selection ranging from 400 to 500 bp. Quantity of final libraries was measured using Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific) and the library size was determined using Agilent 2200 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Among the constructed sequencing libraries, 29 libraries were sequenced with the HiSeq. 2500 (Illumina Inc.) as 100 bp single-end reads and remaining one library for S. tsukubaensis was sequenced with the Miseq v.2 (Illumina Inc.) with 50 bp single-read recipe. Finally, 0.46 to 5.18 Gbp of raw sequence data were obtained and the read qualities were examined by creating sequencing QC reports function of CLC genomic workbench version 6.5.1 (CLC bio, Denmark) (Online-only Table 1 and Fig. 1a).Fig. 1Quality of the genome sequencing data. (a) Distribution of Illumina reads quality based on Phred score. (b) Read quality distribution of PacBio reads. Black line indicates total number of bases in the reads which have greater read quality than the corresponding read quality value on x-axis.Long-read (PacBio) genome sequencingA total of 5 μg gDNA was used as input for PacBio genome sequencing library preparation. The sequencing library was constructed with the PacBio SMRTbellTM Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA) following manufacturer’s instructions. Fragments smaller than 20 kbp were removed using the Blue Pippin Size selection system (Sage Science, Beverly, MA, USA) and the constructed libraries were validated using Agilent 2100 Bioanalyzer (Agilent Technologies). Final SMRTbell libraries were sequenced using one or two SMRT cells with P6-C4-chemistry (DNA Sequencing Reagent 4.0) on the PacBio RS II sequencing platform (Pacific Biosciences). Approximately, 0.5 to 3.0 Gbp of raw sequence data were generated (Online-only Table 1).Genome assemblyAmong the raw PacBio sequencing reads, only the reads with a read quality value greater than 0.75 and a length longer than 50 bp were filtered (Fig. 1b). Post filtered reads were assembled by the hierarchical genome assembly process workflow (HGAP, Version 2.3), including consensus polishing with Quiver18. For each assembled contig, error correction was performed based on their estimated genome size and average coverage. Raw reads from the Illumina sequencing were quality trimmed using CLC genomic workbench version 6.5.1 (ambiguous limit 2 and quality limit 0.05) and assembled using de novo assembly function of CLC genomic workbench version 6.5.1 with default parameters. To expand the assembled contigs, all of assembled PacBio and Illumina contigs were aligned using MAUVE 2.4.019 and linked using GAP5 program (Staden package)20.Genome correctionQuality trimmed Illumina sequencing reads were mapped to the assembled genome using CLC genomic workbench version 6.5.1 (mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.9, and similarity fraction 0.9). Conflicts showing more than 80% frequency for Illumina reads were corrected as Illumina sequence (Table 1). In addition, percentage of mapped Illumina reads on to the assembled genome represents degree of completeness (Table 1 and Fig. 2b). Completeness of gene space was estimated using the BUSCO v3 (Table 2)21.Table 1The statistics of genome assembly and correction.No.SpeciesFinal scaffolds (No.)Scaffold length before correction (bp)Mapped Illumina reads (%)Conflict positions (No.)Added bases (No.)Deleted bases (No.)Scaffold length after correction (bp)G + C contets (%)Assembly accession number1Streptomyces clavuligerus26,748,589 and 1,795,49671.16 and 14.037436,748,591 and 1,795,49572.5GCA_005519465.12Streptomyces tsukubaensis17,963,72795.13151507,963,74271.9GCA_003932715.13Streptomyces galilaeus17,756,17690.565134167,756,19471.4GCA_008704575.14Streptomyces nitrosporeus17,581,54393.505135167,581,56272.2GCA_008704555.15Streptomyces subrutilus17,604,70596.4128626907,604,97473.4GCA_008704535.16Streptomyces viridosporus T7A17,280,44790.44908907,280,53672.6GCA_008704515.17Streptomyces kanamyceticus110,133,52599.09376375310,133,89771.0GCA_008704495.18Streptomyces aureofaciens17,757,87384.8616957,757,87772.6GCA_008704475.19Streptomyces prasinus17,646,57689.701,0251,02157,647,59272.0GCA_008704445.110Streptomyces fradiae16,725,57497.635506,725,57974.7GCA_008704425.111Streptomyces alboniger17,962,59499.1219319317,962,78671.2GCA_008704395.112Streptomyces coeruleorubidus19,334,39999.671,2971,29909,335,69871.1GCA_008705135.113Streptomyces cinereoruber17,516,47499.7417817807,516,65272.9GCA_009299385.114Streptomyces nodosus17,772,56499.51262527,772,58770.9GCA_008704995.115Streptomyces vinaceus17,673,32992.4618018007,673,50972.3GCA_008704935.116Streptomyces platensis18,500,67399.75354352138,501,01271.1GCA_008704855.117Streptomyces spectabilis19,806,22295.3093493809,807,16072.4GCA_008704795.118Streptomyces chartreusis19,911,63798.4246146109,912,09871.0GCA_008704715.119Stretpomyces rimosus19,361,13296.22222209,361,15472.0GCA_008704655.120Streptomyces albofaciens24,757,761 and 4,494,33653.36 and 45.5350450134,757,978 and 4,494,61772.3GCA_008634025.121Streptomyces filamentosus25,742,252 and 2,129,92875.22 and 24.283,2183,22815,744,022 and 2,131,38573.6GCA_008634015.122Streptomyces venezuelae ATCC 1071218,223,43999.849681158,223,50572.5GCA_008639165.123Streptomyces venezuelae ATCC 2111317,893,62299.8517318107,893,80372.5GCA_008639045.124Streptomyces venezuelae ATCC 1059517,871,44995.50353437,871,48072.5GCA_008705255.125Streptomyces venezuelae ATCC 1506818,557,61599.7158758708,558,20271.9GCA_008642375.126Streptomyces venezuelae ATCC 1458318,018,46187.17292748,018,48471.3GCA_008642355.127Streptomyces venezuelae ATCC 1458418,941,82399.0025525508,942,07871.2GCA_008642315.128Streptomyces venezuelae ATCC 1458518,048,13982.346441268,048,15471.3GCA_008642335.129Streptomyces venezuelae ATCC 2178217,525,23590.50878707,525,32271.9GCA_008642295.130Streptomyces venezuelae ATCC 2101817,746,21491.61595747,746,26772.1GCA_008642275.1Fig. 2Genome assembly of 30 streptomycetes. (a) Strategy for genome assembly and corrections. (b) Profile of Illumina reads mapped on assembled genomes. Data were visualized using SignalMap (Roche NimbleGen, Inc.). Red line indicates the average Illumina read coverage of all genomic positions.Table 2Gene space completeness of completed genomes.No.SpeciesComplete and single-copyComplete and duplicatedFragmentedMissingTotalGene space completeness (%)1Streptomyces clavuligerus34300935297.42Streptomyces tsukubaensis35000235299.43Streptomyces galilaeus35100135299.74Streptomyces nitrosporeus352000352100.05Streptomyces subrutilus34900335299.16Streptomyces viridosporus T7A35100135299.77Streptomyces kanamyceticus352000352100.08Streptomyces aureofaciens35000235299.49Streptomyces prasinus35000235299.410Streptomyces fradiae35100135299.711Streptomyces alboniger35100135299.712Streptomyces coeruleorubidus35100135299.713Streptomyces cinereoruber35100135299.714Streptomyces nodosus35001135299.415Streptomyces vinaceus34901235299.116Streptomyces platensis35100135299.717Streptomyces spectabilis35001135299.418Streptomyces chartreusis35100135299.719Stretpomyces rimosus35100135299.720Streptomyces albofaciens34640235299.421Streptomyces filamentosus35100135299.722Streptomyces venezuelae ATCC 10712352000352100.023Streptomyces venezuelae ATCC 21113352000352100.024Streptomyces venezuelae ATCC 10595352000352100.025Streptomyces venezuelae ATCC 1506835100135299.726Streptomyces venezuelae ATCC 1458335100135299.727Streptomyces venezuelae ATCC 1458435100135299.728Streptomyces venezuelae ATCC 1458535100135299.729Streptomyces venezuelae ATCC 2178234900335299.130Streptomyces venezuelae ATCC 2101835000235299.4Genome annotation and secondary metabolite biosynthetic gene cluster predictionThe complete genome sequences of streptomycetes were submitted to the NCBI GenBank database and annotated by the latest updated version of NCBI Prokaryotic Genome Annotation Pipeline (PGAP)22. Using the GenBank formatted files of each genomes as input, secondary metabolite biosynthetic gene clusters were predicted by antiSMASH 4.023.Data RecordsRaw reads from short-read (Illumina) and long-read (PacBio) sequencing were deposited in the NCBI Sequence Read Archive (SRA) (Online-only Table 1)24,25. 30 complete genome sequences were deposited in GenBank via the NCBI’s submission portal (Table 3)26–55. Detailed information on the predicted 922 smBGCs in 30 streptomycetes genomes has been deposited in FigShare56.Table 3Summary of genome annotation.No.SpeciesCDS (No.)16s rRNA (No.)tRNA (No.)Genome accession numberBioProject accession number1Streptomyces clavuligerus6,8801866CP027858PRJNA4141362Streptomyces tsukubaensis6,3761866CP020700PRJNA3820163Streptomyces galilaeus6,7251876CP023703PRJNA4122924Streptomyces nitrosporeus6,3641874CP023702PRJNA4122925Streptomyces subrutilus6,4312168CP023701PRJNA4122926Streptomyces viridosporus T7A6,2111870CP023700PRJNA4122927Streptomyces kanamyceticus8,3841866CP023699PRJNA4122928Streptomyces aureofaciens6,4533371CP023698PRJNA4122929Streptomyces prasinus6,2631868CP023697PRJNA41229210Streptomyces fradiae5,4651865CP023696PRJNA41229211Streptomyces alboniger6,6131867CP023695PRJNA41229212Streptomyces coeruleorubidus8,0581867CP023694PRJNA41229213Streptomyces cinereoruber6,3921869CP023693PRJNA41229214Streptomyces nodosus6,4911868CP023747PRJNA41229215Streptomyces vinaceus6,6032168CP023692PRJNA41229216Streptomyces platensis7,0322167CP023691PRJNA41229217Streptomyces spectabilis8,2121865CP023690PRJNA41229218Streptomyces chartreusis8,3961871CP023689PRJNA41229219Stretpomyces rimosus7,7562168CP023688PRJNA41229220Streptomyces albofaciens7,5202167PDCM00000000PRJNA41229221Streptomyces filamentosus6,8322470PDCL00000000PRJNA41229222Streptomyces venezuelae ATCC 107127,3772167CP029197PRJNA45454723Streptomyces venezuelae ATCC 211136,9872167CP029196PRJNA45454724Streptomyces venezuelae ATCC 105956,9422167CP029195PRJNA45454725Streptomyces venezuelae ATCC 150687,7002169CP029194PRJNA45454726Streptomyces venezuelae ATCC 145837,1541866CP029193PRJNA45454727Streptomyces venezuelae ATCC 145847,8321865CP029192PRJNA45454728Streptomyces venezuelae ATCC 145857,0961866CP029191PRJNA45454729Streptomyces venezuelae ATCC 217826,6551869CP029190PRJNA45454730Streptomyces venezuelae ATCC 210186,7692171CP029189PRJNA454547Technical ValidationStreptomyces have drawn considerable attention because of their ability to produce various clinically important secondary metabolites. Total 30 streptomycetes genomes were sequenced by using both PacBio and Illumina sequencing methods to elucidate their biosynthetic potential. After cleaning the reads, on average 98,380 PacBio reads with 11,725 bp length and 18,223,235 Illumina reads with 100 bp length (50 bp for S. tsukubaensis) were generated (Fig. 1a,b and Online-only Table 1). Through the assembly of reads from two sequencing platforms using HGAP, CLC workbench, MAUVE, and GAP5 programs, single linear scaffolds ranging from 6.7 to 10.1 Mbp in length with 72% G + C contents were obtained for 27 streptomycetes, whereas two scaffolds were finally constructed for three remaining streptomycetes, S. clavuligerus (6.7 and 1.8 Mbp), S. albofaciens (4.8 and 4.5 Mbp), and S. filamentosus (5.7 and 2.1 Mbp) (Table 1). S. clavuligerus has been reported to have a large linear plasmid with a length of 1.8 Mbp, so the genome was correctly assembled into a single chromosome, while the S. albofaciens and S. filamentosus genomes appear to be assembled into two divided scaffolds11,57. To increase the accuracy of the assembled genome sequences, Illumina sequences showing more than 80% coverage at the conflict sites were taken as the corrected ones (Table 1). Approximately, 96.32% of Illumina sequencing reads were successfully mapped to the corresponding genomes (Table 1 and Fig. 2b). The completeness of the genomes were assessed using the BUSCO approach with a total of 352 orthologue groups from the Actinobacteria Dataset21. Results showed that 29 genomes have more than 99.1% gene space completeness and the S. clavuligerus genome has 97.4% gene space completeness (Table 2). Following NCBI PGAP, 30 genomes were annotated with 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs on average (Table 3). Finally, based on the annotation, a total of 922 smBGCs were predicted in 30 streptomycetes genomes (Fig. 3). Detailed information, such as genomic positions, types, and putative products of each smBGC are publicly available in Figshare56.Fig. 3Secondary metabolite biosynthetic gene clusters in 30 complete streptomycetes genomes.