BACKGROUND
actinomycetes are gram-positive mycelial bacteria found predominantly in soil, but they also occur in symbiotic associations with terrestrial and aquatic invertebrates.
they undergo a complex process of morphological and physiological differentiation that leads to the production of exospores and specialised metabolites  <cit>  with a wide range of biological activities.
while the function of many of these molecules in the natural environment is not always evident, they are believed to provide a competitive advantage to the producing organism  <cit> .
many of these specialised metabolites possess potent antibiotic activity, and actinomycetes produce over 70 % of the natural product scaffolds found in clinically used anti-infective agents  <cit> .
the pioneering sequencing and analysis of the genomes of streptomyces coelicolor a <dig>  <cit>  and streptomyces avermitilis  <cit>  revealed that actinomycetes possess an unexpected abundance of natural product biosynthetic gene clusters and thus that they have the potential to make many more compounds than previously thought.
this led to the “genome mining” approach to natural product discovery, where bioinformatic analysis is used to estimate the biosynthetic capacity, and potential metabolite novelty, of an organism before extensive analysis in the laboratory.
genome mining of actinomycetes and other microorganisms has already provided access to many novel biosynthetic pathways and metabolites that otherwise would have remained undetected  <cit> .
it may be particularly useful when analysing novel and possibly difficult to culture microorganisms isolated from unusual and unexploited habitats such as the oceans, deserts, and the surfaces of plants and animals.
to be carried out efficiently, genome mining relies on the availability of a good quality genome sequence obtained at an affordable price and in a short time-frame.
until recently, whole genome shotgun sequencing using next-generation sequencing  technologies could not be expected to yield less than several hundred contigs for an actinomycete genome of  <dig> – 12 mb, with biosynthetic gene clusters often split over several contigs.
much bioinformatic analysis and contig-stitching by, for example, pcr would often be required to identify a complete cluster.
moreover, the short read length of second generation sequencing technologies  makes it very difficult to correctly assemble the long repetitive sequences typically found in biosynthetic gene clusters containing modular polyketide synthases  or non-ribosomal peptide synthetases .
in such clusters, it is not unusual to find regions of high homology between genes and intragenic tandem repeats of 650-1000 bp with close to 100 % nucleotide sequence identity, well beyond the read length provided by illumina ; e.g., the s. coelicolor coelimycin pks gene sco <dig>  positions 3879- <dig> and 11986- 12639; and the calcium-dependent antibiotic nrps gene sco <dig>  positions 13187- <dig> and 16307- <dig>  the long sequence reads provided by third generation sequencing techniques such as the pacific biosciences smrt technology should allow more reliable assemblies of pks and nrps gene clusters and, in principle, yield single contigs for all of the replicons present in an actinomycete.
the smrt technology also produces more even sequence coverage of the high mol% g + c dna found in actinomycete genomes than other ngs platforms  <cit> .
streptomyces leeuwenhoekii  was isolated, together with many other novel actinomycetes, from the saline chaxa lagoon in the high-altitude atacama desert in northern chile  <cit> .
it produces previously described metabolites , but also novel polyketide antibiotics, the chaxamycins and chaxalactins.
chaxamycin a – d are four novel ansamycin-type polyketides with promising antibacterial activity against mrsa and anti-proliferative activity resulting from the inhibition of the atpase activity of the human hsp <dig> protein  <cit> .
a draft genome sequence of this strain with  <dig> contigs had been derived previously from illumina ga iix 100 bp paired-end reads  <cit> ; attempts to mine this sequence for the chaxamycin and chaxalactin biosynthetic gene clusters  revealed many inconsistencies and misassemblies , presumably a consequence of the short read lengths of the illumina technology and the repetitive nature of the two gene clusters.
given the unusual origin of s. leeuwenhoekii and our desire to analyse its biosynthetic potential, we set out to sequence its genome using the most advanced technology available.
this has allowed us to generate an almost complete chromosome sequence as well as the sequences of two plasmids as single contigs without recourse to gap-closing or sequencing of clones from a genomic library; to our knowledge, this is the first time that this has been achieved with an actinomycete.
we also report here our findings on the advantages and limitations of the two technologies we used, illumina miseq and pacific biosciences rsii, and we propose a pipeline for the generation of high quality actinomycete genome sequences.
RESULTS
availability of data
the fully annotated sequences presented in this work have been deposited in the european nucleotide archive under study accession number prjeb <dig> .
the sequences of psle <dig>  psle <dig> and the chromosome have been assigned accession numbers ln <dig>  ln <dig> and ln <dig> respectively.
sequencing and assembly of the s. leeuwenhoekii genome
two technologies were used to sequence genomic dna isolated from s. leeuwenhoekii: illumina miseq  and pacific biosciences  rsii .
assembly of the illumina sequencing data yielded  <dig> contigs, assembled into  <dig> scaffolds, totalling 8064420 bp.
assembly of the pacbio sequencing data produced three contigs of  <dig>   <dig> and 94746 bp, totalling 8000192 bp.
the pacbio contig of  <dig>  mb was expected to contain most of the sequence of the chromosome, and was referred to as c34-chromosome version  <dig>  the small 9613 bp contig from the pacbio assembly was found to match the  <dig>  mb contig from position  <dig> to  <dig>  but with only 92 % identity, possibly indicating that it originated from reads with accumulated errors.
these small, error-prone, contigs have been observed by others using pacbio sequencing .
this contig was discarded from any further analysis.
a detailed description of the sequencing and bioinformatic analysis of this and following sections can be found in additional file  <dig> – materials and methods.
correction of the pacbio  <dig>  mb contig  using the illumina assembly
comparison of the illumina miseq and pacbio assemblies readily identified problems of misassembly in the illumina contigs, and in particular in the regions containing polyketide biosynthetic gene clusters, a particular focus of our initial interest in s. leeuwenhoekii .
however, aware of the higher nucleotide-accuracy of the illumina technology compared to pacbio, we mapped the illumina miseq contigs  over the pacbio-generated c34-chromosome version  <dig>  the generated alignment was manually edited with gap <dig>  <cit>  to correct the pacbio sequence with the illumina contigs while accommodating possible misassembles and systematic errors inherent in the illumina technology  <cit> .
most of the differences were apparent omissions in the pacbio sequence of a c or g in homopolymeric runs of three or more cs or gs.
these omissions were confirmed by analysis of individual sequence differences; the missing nucleotides in the pacbio sequence resulted in frame shifts that were readily identified using gc-frame plot  <cit>  in artemis  <cit>  and substantiated by inspection of the corresponding amino acid sequences.
our analysis indicated that the illumina sequence always contained the correct number of bases in these homopolymeric stretches, and consequently it was used to correct the pacbio assembly.
this resulted in the insertion of  <dig> bases and an additional  <dig> base changes, a total of  <dig> corrections in a final chromosomal assembly of  <dig> bases .
this sequence was referred to as c34-chromosome version  <dig>
extension of  <dig>  mb corrected-contig  with the illumina assembly
during the previous correction step we identified sequence in the illumina assembly that was absent from the pacbio assembly.
this was achieved by using all of the illumina assembled data instead of the illumina reads, which is the usual bioinformatic practice .
we found that illumina contig  <dig>  mapped at the 5’-end of the pacbio  <dig>  mb contig but contained  <dig>  kb of additional sequence .
after careful examination of the genetic content of contig  <dig>  we concluded that the extra  <dig>  kb was genuine s. leeuwenhoekii sequence likely located at the end of the terminal inverted repeat .
blast analysis  <cit>  and alignment in gap <dig> were then used to extend the 5’-end of the chromosomal sequence with an extra  <dig> bases to yield a chromosome of 7903888 bp, referred to as c34-chromosome version  <dig>
further correction of c34-chromosome version  <dig> with the illumina paired-end reads
the original, unassembled, illumina paired-end reads with quality values were aligned to version  <dig> of the chromosome with two different programs, bwa  <cit>  and bowtie  <dig>  <cit> , processed with samtools and bcftools  <cit>  to call the potential variants, and these were then studied within gap <dig>  we focused on the additions/omissions reported by both alignment programs.
while we decided not to incorporate any of the reported base changes, we did correct a few additions/omissions, in particular an erroneous addition at position  <dig> that was only reported by the bwa alignment  which repaired a reading-frame shift in a polyketide synthase gene that is part of the chaxalactin biosynthetic gene cluster .
three corrections were also made at the beginning of the proposed terminal inverted repeat  at the 5’-end of the sequence; a c was added in a run of five cs after the g at position  <dig>  giving six cs; a c was added after a at new position  <dig> ; and a c was added after a at new position  <dig> .
finally, a g was added at position  <dig> , correcting an omission reported by both programs that introduced a frame shift in an uncharacterised polyketide synthase gene .table  <dig> putative biosynthetic gene clusters for specialised metabolites
not identified
160425
189028
not identified
684373
654830
t1pks-terpene
1211049
1289829
terpene
3056325
3058819
siderophore
5237176
5244356
not identified
psle <dig> 103389
psle <dig> 105999
the final chromosomal sequence, referred to as c34-chromosome version  <dig>  contains  <dig> nucleotides and has a mol% g + c content of  <dig>  %.
annotation of the streptomyces leeuwenhoekii chromosome
annotation of the s. leeuwenhoekii chromosome version  <dig> was performed using prodigal  <cit>  to identify protein coding sequences  followed by basys  <cit>  for assignment of putative function.
we chose gene identifiers starting with “sle”, for “streptomyces leeuwenhoekii”, followed by five digits starting with “sle_00010” with increments of  <dig> to allow for the addition of subsequently identified genes and genetic features.
neither prodigal nor basys annotated rrna or trna genes, so the chromosome sequence was submitted to the rast server  <cit>  and the rrna and trna annotations added to generate the published s. leeuwenhoekii chromosome sequence.
identification of the possible terminal inverted repeats  and chromosome ends
streptomyces species frequently possess linear chromosomes with terminal inverted repeats  of almost identical sequence with covalently-bound terminal proteins for priming of replication  <cit> .
the length of the tir is very variable among species, ranging from only 14 bp in streptomyces hygroscopicus  <dig>  <cit>  to over  <dig> mbp in s. coelicolor  <cit> .
blast analysis and examination in artemis revealed that the last  <dig> bases at the 3’-end of the chromosome sequence  were 99 % identical to a segment of reverse-complementary sequence around position 388 kb .
it is likely that this 7 kb sequence at the right end represents the start of the right tir, and was acquired by some of the long pacbio reads that extended from the region next to the tir, while the rest of the reads from the right tir have probably been assembled with the left tir.
visualisation of the pacbio assembly bam file revealed a pronounced increase in coverage of the first 380 kb of the assembly , probably reflecting the incorporation of reads from both the left and right tir in the assembly of the left tir.fig.
<dig> schematic representation of the s. leeuwenhoekii chromosome, circular plasmid psle <dig>  and linear plasmid psle <dig> .
the chromosome is represented as an open circle, covering only the published sequence without the duplication of the terminal inverted repeat .
from outside to inside, the concentric circles represent: nucleotide position; protein coding sequences  on the forward strand; pcss on the reverse strand; pcss for putative biosynthetic genes for specialised metabolites ; the orange box shown in the fifth circle indicates the chaxamycin biosynthetic gene cluster; trna and rrna genes are shown in the sixth and seventh lines, respectively, in dark blue; the eighth concentric circle shows the gc-plot  and the inner-most circle the gc-skew /] window size = 10000; base step size = 200), both calculated using the sequence with both tirs, a window size of  <dig> and a step size of  <dig> .
for psle <dig> and psle <dig>  pcss are coloured red for putative regulatory genes; green, for plasmid replication and partitioning genes; the fourth circle in shows the gc-plot and the inner-most circle the gc-skew, both calculated as for the chromosome.
for psle <dig>  phage-related genes are shown in orange, and the type iii pks  gene is shown in brown.
for psle <dig>  genes with known plasmid functions are in orange; genes annotated as mobile elements and involved in transposition are in pink; the lasso-peptide biosynthetic gene cluster is shown in dark orange.
not to scale
fig.
<dig> sequencing and assembly pipeline.
the sequencing and assembly pipeline followed in this work  and suggested as strategy for actinomycete genome sequencing
detailed analysis of the end of the left tir  identified two genes, sle_ <dig> and sle_ <dig>  with 84 % nucleotide sequence identity to each other, and predicted amino acid sequences that were 73 % and 72 % identical, respectively, to a helicase-like protein encoded by sco <dig> of s. coelicolor, homologues of which are present and highly conserved at both ends of all sequenced linear replicons  from streptomyces species  <cit> .
the presence of two contiguous helicase-like encoding genes has not been described before but detailed sequence inspection failed to reveal possible misassembly.
mfold  <cit>  analysis of the dna sequence upstream of sle_ <dig>  revealed palindromic repeats with the potential to form a complex secondary structure  similar to those found in other streptomyces linear replicons  <cit> .
our analysis suggests that the tirs of the s. leeuwenhoekii chromosome extend for about 388 kb, at the upper end of published streptomyces tir sequences.
this also implies that the hygromycin a biosynthetic gene cluster, located between position 160-189 kb, could be duplicated at each end of the chromosome should both tirs show complete gene conservation.
while still infrequent, biosynthetic gene clusters for specialised metabolites localised in the tirs of actinomycetes have been described previously  <cit>  and demonstrated experimentally  <cit> .
assembly and annotation of psle <dig>  an  <dig>  kb circular plasmid
the 94746 bp pacbio-contig contained direct repeats of  <dig>  kb present at both ends, indicative of an 86 kb circular dna molecule.
most of this sequence was also found in a contig of 86370 bp in the illumina assembly.
blast analysis using the non-redundant ncbi database suggested that it represents a plasmid, which we called psle <dig>
a detailed description of the assembly of the circular dna sequence using both pacbio and illumina data is given in the additional file  <dig> and additional file  <dig>  psle <dig> possesses a base composition of 69 % mol% g + c.  <dig> putative pcss were identified with prodigal and tagged as “sle2_001”; putative functions were added manually in artemis using blast and pfam  <cit>  searches.
for most of the pcss, in particular those encoding putative phage particle proteins, the annotation was based on that of pzl <dig>  <cit> , a well characterised plasmid with high levels of homology and synteny to psle <dig>  genes encoding para and parb partitioning proteins were identified with pfam, supporting the proposition that this contig represents an autonomously replicating element.
psle <dig> para shows no discernible identity to the chromosomal para, but is 62 % identical to pzl <dig> c para and 67 % identical to the putative plasmid partitioning protein para from streptomyces rochei .
psle <dig> parb shares 31 % identity with the chromosomal parb, but 41 % identity with pzl <dig> c parb and 67 % identity with the putative plasmid partitioning protein parb from s. rochei .
interestingly, psle <dig> contains two genes encoding putative integrases, suggesting that the plasmid might be capable of insertion into the chromosome.
a search with antismash  <cit>  did not identify any putative specialised metabolite gene clusters in psle <dig> .
assembly and annotation of psle <dig>  a >132 kb linear plasmid
two illumina contigs, contig <dig> with 97005 bp and contig <dig> with 32697 bp, did not match any sequence in the pacbio assembly.
blast searches against the non-redundant ncbi database indicated that it could represent a plasmid, which we designated psle <dig>  we were subsequently able to join the illumina contigs using corrected pacbio reads to yield a 132 kb sequence .
psle <dig> has a base composition of 70 mol% g + c. pcss were identified using prodigal and tagged as “sle2_001”; putative functions were manually annotated in artemis using blast and pfam searches.
we could not identify a clear direct repeat indicative of a circular plasmid, as we did for psle <dig>  or a putative tir characteristic of a linear plasmid, suggesting that some sequence might be missing.
however, the 5’-end of psle <dig>  is highly similar  to the end of the tir identified for the chromosome , and includes a gene for a putative terminal helicase  with 87 % and 85 % amino acid identity to sle_ <dig> and sle_ <dig>  respectively.
such high identity between linear chromosomes and linear plasmids is a feature of streptomyces species  <cit>  and suggests that psle <dig> is a linear plasmid and that this sequence is at one end of a tir.
two contiguous genes were found by pfam searches to encode possible functional homologues of the partition proteins para and parb .
pulse-field gel-electrophoresis  of total dna isolated from s. leeuwenhoekii revealed an extrachromosomal replicon with an apparent linear size of between  <dig> and  <dig>  kb , consistent with the predicted size of psle <dig>
as for psle <dig>  we found a gene, sle2_ <dig>  encoding a putative integrase, suggesting that the plasmid might be capable of integrating into the chromosome.
in addition, we identified at least three genes encoding possible conjugation functions  suggesting that the plasmid might be capable of self-transmission by conjugation.
an interesting feature is the presence of a gene, sle2_ <dig>  encoding a putative zeta-toxin  e-value  <dig> e-45) potentially involved in ensuring the maintenance and segregation of the plasmid  <cit> ; we did not find a gene encoding a putative anti-toxin, but we did find a toxin-antitoxin system encoded in the chromosome similar to that reported for s. coelicolor and streptomyces lividans  <cit> .
antismash did not reveal any putative natural product gene clusters, but we did find a gene cluster potentially encoding the biosynthesis of a lasso-peptide, which we have subsequently identified and characterised .
intriguingly, there are several genes encoding putative transposases, many with frame shifts .
this region was not present in the illumina assembly, and lies between illumina contigs  <dig> and 0079; only the pacbio corrected-reads contained the full sequence .
the presence of many repeated sequences with high levels of nucleotide sequence identity may explain their absence from the illumina assembly.
general characteristics of the genome sequence
our assembled chromosome sequence contains 7903895 bp, with a mol% g + c content of 73 %, consistent with other members of the genus streptomyces.
with the proposed addition of the right hand 388 kb tir, the predicted size of the genome would be 8285171 bp .
this is similar to the size of many streptomycete genomes .
<dig> pcss were predicted for our assembled sequence, and  <dig> pcss if we include the pcss predicted for the right tir.
a table of cog  functional categories  <cit>  was determined .table  <dig> cog functional categories.
cog  functional categories of chromosomal protein codding sequences identified in s. leeuwenhoekii chromosome, and from s. coelicolor for comparison
streptomyces leeuwenhoekii
streptomyces coelicolor
the s. leeuwenhoekii chromosome contains six rrna operons located between nucleotide positions  <dig> and 6687647; interestingly, an orphan gene encoding a 5s rrna was found at position  <dig>  dnaa and dnan replication proteins are encoded in genes spanning nucleotide positions 4260071- <dig> and 4256851- <dig>  respectively, indicating that the likely origin of replication of the chromosome lies between  <dig> and  <dig>  consistent with precedent, the two rrna operons that lie between nucleotide positions  <dig> and  <dig>  are located on the complementary strand, while the four rrna operons located between nucleotide positions  <dig> and  <dig> are located on the top strand .
we identified  <dig> trna genes , similar to the number found in other streptomyces .
the general characteristics of the s. leeuwenhoekii genome are summarised in table  <dig> table  <dig> general characteristics of the s. leeuwenhoekii genome
biosynthetic gene clusters for specialised metabolites
putative gene clusters encoding the biosynthesis of specialised metabolites were identified with antismash  <dig>  <cit>  and non-automated searches with blast and pfam .
s. leeuwenhoekii was known to produce the siderophore desferrioxamine e and the antibiotic hygromycin a  <cit> ; we identified probable biosynthetic gene clusters for both of these metabolites by searching for homologues of the corresponding gene clusters from s. coelicolor and streptomyces hygroscopicus nrrl  <dig>  respectively.
the desferrioxamine e gene cluster extends from sle_ <dig> to sle_ <dig> and shows complete gene synteny with the homologous gene cluster from s. coelicolor with protein identities varying between 77 % and 93 %, while the putative hygromycin a gene cluster, which spans from sle_ <dig> to sle_ <dig>  shows almost complete synteny with the homologous gene cluster from s. hygroscopicus, with protein identities of 64 % to 90 %.
interestingly, antismash did not identify the hygromycin a gene cluster which lies within the proposed left tir; two putative type-i pks gene clusters and one putative hybrid type-i pks-nrps gene cluster were also found in the left tir.
if, as we have proposed, the tirs span for 388 kb, then all four of these gene clusters would be duplicated at the other end of the chromosome.
s. leeuwenhoekii is also known to produce two novel families of polyketide antibiotics, the chaxalactins and the ansamycin-like chaxamycins  <cit> .
the biosynthetic gene clusters for both antibiotics were identified and are currently under investigation.
we also identified three putative gene clusters each encoding the biosynthesis of unknown lasso-peptides .
two of them, the “lasso-peptide 1” and “lasso-peptide 2” gene clusters  are located on the chromosome, while the “lasso-peptide 3” gene cluster  is located on the linear plasmid psle <dig>  all three gene clusters are currently under study in our laboratory, where pcr amplification and sanger sequencing have confirmed that the assembly and sequence reported here is correct.
these clusters were not identified by antismash.
CONCLUSIONS
benefits and challenges of sequencing technologies: a revised pipeline
to our knowledge, this is the first report of the use of ngs to produce a high quality and non-fragmented genome sequence of an actinomycete, an essential prerequisite for efficient genome mining for natural product discovery in these gc-rich bacteria.
our assembly yielded the sequences of a single chromosomal contig, a complete circular plasmid , and most of a linear plasmid .
although the illumina miseq assembly produced a single contig for psle <dig>  it would not have been possible to confirm its circularity and completeness without the pacbio data.
overall, we used two smrt cells  resulting in over 120x coverage.
thus, with the improving capacity of the pacbio smrt cells, we predict that a maximum of three smrt cells should be sufficient to obtain the entire complement of replicons of an actinomycete genome of around 8 mb in single contigs.
this contrasts with the previously published illumina draft genome sequence of s. leeuwenhoekii  <cit>  that contained  <dig> contigs in which we found many misassemblies and missing sequence .
the pacbio long reads also permitted the identification of the start of the right hand tir, but they did not provide any sequence information for the 5 kb at the ends of the chromosome and the linear plasmid psle <dig>  which was obtained from the illumina data.
the reason for this is unknown, but it could reflect the different strategies used for library construction; while pacbio focuses on large fragments of ~20 kb, the average size of the illumina library used for sequencing was 550 bp.
also, no specific procedure was used during dna purification to remove the protein that is covalently bound to the termini of the linear replicons, perhaps resulting in lower sequence coverage of the terminal regions that might be further exacerbated by the large fragment size used for pacbio sequencing.
surprisingly, the pacbio assembly lacked the linear psle <dig> sequence, although the sequence information was present in the corrected pacbio reads.
the illumina assembly contained two large contigs covering most of psle <dig> that were merged into a contiguous sequence using the pacbio corrected reads, but this plasmid would have not been identified with the pacbio assembly alone.
these two findings highlight the importance of the current need to use both pacbio and illumina assemblies, instead of assembling only the pacbio data and then using the illumina reads to correct the assembly, which appears to be the accepted practice  <cit> .
we cannot explain the lack of assembly of some of the pacbio data present in the corrected reads  but this was compensated for by using the illumina miseq assembly.
in summary, for a de novo shotgun genome sequence from an actinomycete aimed at yielding single contigs per replicon, we currently propose a strategy  that includes sequencing genomic dna with pacbio rsii using initially two  smrt cells and a >20 kb insert library  combined with illumina miseq paired-end sequencing of a 500 bp library without pcr amplification ).
both data sets are assembled and compared, and the illumina contigs used to correct the pacbio nucleotide omissions/additions, which should be confirmed using gc frame plot and blast analyses.
this consensus is further corrected with the illumina reads.
despite the highly efficient current assembly algorithms, a considerable amount of human input was still needed to obtain a high quality single contig assembly, and accurate annotation of gene function.

 actinomycetes are gram-positive mycelial bacteria found predominantly in soil, but they also occur in symbiotic associations with terrestrial and aquatic invertebrates. they undergo a complex process of morphological and physiological differentiation that leads to the production of exospores and specialised metabolites with a wide range of biological activities. while the function of many of these molecules in the natural environment is not always evident, they are believed to provide a competitive advantage to the producing organism. many of these specialised metabolites possess potent antibiotic activity, and actinomycetes produce over 70 % of the natural product scaffolds found in clinically used anti-infective agents. the pioneering sequencing and analysis of the genomes of streptomyces coelicolor a and streptomyces avermitilis revealed that actinomycetes possess an unexpected abundance of natural product biosynthetic gene clusters and thus that they have the potential to make many more compounds than previously thought. this led to the “genome mining” approach to natural product discovery, where bioinformatic analysis is used to estimate the biosynthetic capacity, and potential metabolite novelty, of an organism before extensive analysis in the laboratory. genome mining of actinomycetes and other microorganisms has already provided access to many novel biosynthetic pathways and metabolites that otherwise would have remained undetected. it may be particularly useful when analysing novel and possibly difficult to culture microorganisms isolated from unusual and unexploited habitats such as the oceans, deserts, and the surfaces of plants and animals. to be carried out efficiently, genome mining relies on the availability of a good quality genome sequence obtained at an affordable price and in a short time-frame. until recently, whole genome shotgun sequencing using next-generation sequencing technologies could not be expected to yield less than several hundred contigs for an actinomycete genome of – 12 mb, with biosynthetic gene clusters often split over several contigs. much bioinformatic analysis and contig-stitching by, for example, pcr would often be required to identify a complete cluster. moreover, the short read length of second generation sequencing technologies makes it very difficult to correctly assemble the long repetitive sequences typically found in biosynthetic gene clusters containing modular polyketide synthases or non-ribosomal peptide synthetases. in such clusters, it is not unusual to find regions of high homology between genes and intragenic tandem repeats of 650-1000 bp with close to 100 % nucleotide sequence identity, well beyond the read length provided by illumina; e.g., the s. coelicolor coelimycin pks gene sco positions 3879- and 11986- 12639; and the calcium-dependent antibiotic nrps gene sco positions 13187- and 16307- the long sequence reads provided by third generation sequencing techniques such as the pacific biosciences smrt technology should allow more reliable assemblies of pks and nrps gene clusters and, in principle, yield single contigs for all of the replicons present in an actinomycete. the smrt technology also produces more even sequence coverage of the high mol% g + c dna found in actinomycete genomes than other ngs platforms. streptomyces leeuwenhoekii was isolated, together with many other novel actinomycetes, from the saline chaxa lagoon in the high-altitude atacama desert in northern chile. it produces previously described metabolites, but also novel polyketide antibiotics, the chaxamycins and chaxalactins. chaxamycin a – d are four novel ansamycin-type polyketides with promising antibacterial activity against mrsa and anti-proliferative activity resulting from the inhibition of the atpase activity of the human hsp protein. a draft genome sequence of this strain with contigs had been derived previously from illumina ga iix 100 bp paired-end reads; attempts to mine this sequence for the chaxamycin and chaxalactin biosynthetic gene clusters revealed many inconsistencies and misassemblies, presumably a consequence of the short read lengths of the illumina technology and the repetitive nature of the two gene clusters. given the unusual origin of s. leeuwenhoekii and our desire to analyse its biosynthetic potential, we set out to sequence its genome using the most advanced technology available. this has allowed us to generate an almost complete chromosome sequence as well as the sequences of two plasmids as single contigs without recourse to gap-closing or sequencing of clones from a genomic library; to our knowledge, this is the first time that this has been achieved with an actinomycete. we also report here our findings on the advantages and limitations of the two technologies we used, illumina miseq and pacific biosciences rsii, and we propose a pipeline for the generation of high quality actinomycete genome sequences.  availability of data the fully annotated sequences presented in this work have been deposited in the european nucleotide archive under study accession number prjeb. the sequences of psle psle and the chromosome have been assigned accession numbers ln ln and ln respectively. sequencing and assembly of the s. leeuwenhoekii genome two technologies were used to sequence genomic dna isolated from s. leeuwenhoekii: illumina miseq and pacific biosciences rsii. assembly of the illumina sequencing data yielded contigs, assembled into scaffolds, totalling 8064420 bp. assembly of the pacbio sequencing data produced three contigs of and 94746 bp, totalling 8000192 bp. the pacbio contig of  mb was expected to contain most of the sequence of the chromosome, and was referred to as c34-chromosome version the small 9613 bp contig from the pacbio assembly was found to match the  mb contig from position to but with only 92 % identity, possibly indicating that it originated from reads with accumulated errors. these small, error-prone, contigs have been observed by others using pacbio sequencing. this contig was discarded from any further analysis. a detailed description of the sequencing and bioinformatic analysis of this and following sections can be found in additional file – materials and methods. correction of the pacbio  mb contig using the illumina assembly comparison of the illumina miseq and pacbio assemblies readily identified problems of misassembly in the illumina contigs, and in particular in the regions containing polyketide biosynthetic gene clusters, a particular focus of our initial interest in s. leeuwenhoekii. however, aware of the higher nucleotide-accuracy of the illumina technology compared to pacbio, we mapped the illumina miseq contigs over the pacbio-generated c34-chromosome version the generated alignment was manually edited with gap to correct the pacbio sequence with the illumina contigs while accommodating possible misassembles and systematic errors inherent in the illumina technology. most of the differences were apparent omissions in the pacbio sequence of a c or g in homopolymeric runs of three or more cs or gs. these omissions were confirmed by analysis of individual sequence differences; the missing nucleotides in the pacbio sequence resulted in frame shifts that were readily identified using gc-frame plot in artemis and substantiated by inspection of the corresponding amino acid sequences. our analysis indicated that the illumina sequence always contained the correct number of bases in these homopolymeric stretches, and consequently it was used to correct the pacbio assembly. this resulted in the insertion of bases and an additional base changes, a total of corrections in a final chromosomal assembly of bases. this sequence was referred to as c34-chromosome version extension of  mb corrected-contig with the illumina assembly during the previous correction step we identified sequence in the illumina assembly that was absent from the pacbio assembly. this was achieved by using all of the illumina assembled data instead of the illumina reads, which is the usual bioinformatic practice. we found that illumina contig mapped at the 5’-end of the pacbio  mb contig but contained  kb of additional sequence. after careful examination of the genetic content of contig we concluded that the extra  kb was genuine s. leeuwenhoekii sequence likely located at the end of the terminal inverted repeat. blast analysis and alignment in gap were then used to extend the 5’-end of the chromosomal sequence with an extra bases to yield a chromosome of 7903888 bp, referred to as c34-chromosome version further correction of c34-chromosome version with the illumina paired-end reads the original, unassembled, illumina paired-end reads with quality values were aligned to version of the chromosome with two different programs, bwa and bowtie, processed with samtools and bcftools to call the potential variants, and these were then studied within gap we focused on the additions/omissions reported by both alignment programs. while we decided not to incorporate any of the reported base changes, we did correct a few additions/omissions, in particular an erroneous addition at position that was only reported by the bwa alignment which repaired a reading-frame shift in a polyketide synthase gene that is part of the chaxalactin biosynthetic gene cluster. three corrections were also made at the beginning of the proposed terminal inverted repeat at the 5’-end of the sequence; a c was added in a run of five cs after the g at position giving six cs; a c was added after a at new position; and a c was added after a at new position. finally, a g was added at position, correcting an omission reported by both programs that introduced a frame shift in an uncharacterised polyketide synthase gene.table putative biosynthetic gene clusters for specialised metabolites not identified 160425 189028 not identified 684373 654830 t1pks-terpene 1211049 1289829 terpene 3056325 3058819 siderophore 5237176 5244356 not identified psle 103389 psle 105999 the final chromosomal sequence, referred to as c34-chromosome version contains nucleotides and has a mol% g + c content of  %. annotation of the streptomyces leeuwenhoekii chromosome annotation of the s. leeuwenhoekii chromosome version was performed using prodigal to identify protein coding sequences followed by basys for assignment of putative function. we chose gene identifiers starting with “sle”, for “streptomyces leeuwenhoekii”, followed by five digits starting with “sle_00010” with increments of to allow for the addition of subsequently identified genes and genetic features. neither prodigal nor basys annotated rrna or trna genes, so the chromosome sequence was submitted to the rast server and the rrna and trna annotations added to generate the published s. leeuwenhoekii chromosome sequence. identification of the possible terminal inverted repeats and chromosome ends streptomyces species frequently possess linear chromosomes with terminal inverted repeats of almost identical sequence with covalently-bound terminal proteins for priming of replication. the length of the tir is very variable among species, ranging from only 14 bp in streptomyces hygroscopicus to over mbp in s. coelicolor. blast analysis and examination in artemis revealed that the last bases at the 3’-end of the chromosome sequence were 99 % identical to a segment of reverse-complementary sequence around position 388 kb. it is likely that this 7 kb sequence at the right end represents the start of the right tir, and was acquired by some of the long pacbio reads that extended from the region next to the tir, while the rest of the reads from the right tir have probably been assembled with the left tir. visualisation of the pacbio assembly bam file revealed a pronounced increase in coverage of the first 380 kb of the assembly, probably reflecting the incorporation of reads from both the left and right tir in the assembly of the left tir.fig. schematic representation of the s. leeuwenhoekii chromosome, circular plasmid psle and linear plasmid psle. the chromosome is represented as an open circle, covering only the published sequence without the duplication of the terminal inverted repeat. from outside to inside, the concentric circles represent: nucleotide position; protein coding sequences on the forward strand; pcss on the reverse strand; pcss for putative biosynthetic genes for specialised metabolites; the orange box shown in the fifth circle indicates the chaxamycin biosynthetic gene cluster; trna and rrna genes are shown in the sixth and seventh lines, respectively, in dark blue; the eighth concentric circle shows the gc-plot and the inner-most circle the gc-skew /] window size = 10000; base step size = 200), both calculated using the sequence with both tirs, a window size of and a step size of. for psle and psle pcss are coloured red for putative regulatory genes; green, for plasmid replication and partitioning genes; the fourth circle in shows the gc-plot and the inner-most circle the gc-skew, both calculated as for the chromosome. for psle phage-related genes are shown in orange, and the type iii pks gene is shown in brown. for psle genes with known plasmid functions are in orange; genes annotated as mobile elements and involved in transposition are in pink; the lasso-peptide biosynthetic gene cluster is shown in dark orange. not to scale fig. sequencing and assembly pipeline. the sequencing and assembly pipeline followed in this work and suggested as strategy for actinomycete genome sequencing detailed analysis of the end of the left tir identified two genes, sle_ and sle_ with 84 % nucleotide sequence identity to each other, and predicted amino acid sequences that were 73 % and 72 % identical, respectively, to a helicase-like protein encoded by sco of s. coelicolor, homologues of which are present and highly conserved at both ends of all sequenced linear replicons from streptomyces species. the presence of two contiguous helicase-like encoding genes has not been described before but detailed sequence inspection failed to reveal possible misassembly. mfold analysis of the dna sequence upstream of sle_ revealed palindromic repeats with the potential to form a complex secondary structure similar to those found in other streptomyces linear replicons. our analysis suggests that the tirs of the s. leeuwenhoekii chromosome extend for about 388 kb, at the upper end of published streptomyces tir sequences. this also implies that the hygromycin a biosynthetic gene cluster, located between position 160-189 kb, could be duplicated at each end of the chromosome should both tirs show complete gene conservation. while still infrequent, biosynthetic gene clusters for specialised metabolites localised in the tirs of actinomycetes have been described previously and demonstrated experimentally. assembly and annotation of psle an  kb circular plasmid the 94746 bp pacbio-contig contained direct repeats of  kb present at both ends, indicative of an 86 kb circular dna molecule. most of this sequence was also found in a contig of 86370 bp in the illumina assembly. blast analysis using the non-redundant ncbi database suggested that it represents a plasmid, which we called psle a detailed description of the assembly of the circular dna sequence using both pacbio and illumina data is given in the additional file and additional file psle possesses a base composition of 69 % mol% g + c. putative pcss were identified with prodigal and tagged as “sle2_001”; putative functions were added manually in artemis using blast and pfam searches. for most of the pcss, in particular those encoding putative phage particle proteins, the annotation was based on that of pzl, a well characterised plasmid with high levels of homology and synteny to psle genes encoding para and parb partitioning proteins were identified with pfam, supporting the proposition that this contig represents an autonomously replicating element. psle para shows no discernible identity to the chromosomal para, but is 62 % identical to pzl c para and 67 % identical to the putative plasmid partitioning protein para from streptomyces rochei. psle parb shares 31 % identity with the chromosomal parb, but 41 % identity with pzl c parb and 67 % identity with the putative plasmid partitioning protein parb from s. rochei. interestingly, psle contains two genes encoding putative integrases, suggesting that the plasmid might be capable of insertion into the chromosome. a search with antismash did not identify any putative specialised metabolite gene clusters in psle. assembly and annotation of psle a >132 kb linear plasmid two illumina contigs, contig with 97005 bp and contig with 32697 bp, did not match any sequence in the pacbio assembly. blast searches against the non-redundant ncbi database indicated that it could represent a plasmid, which we designated psle we were subsequently able to join the illumina contigs using corrected pacbio reads to yield a 132 kb sequence. psle has a base composition of 70 mol% g + c. pcss were identified using prodigal and tagged as “sle2_001”; putative functions were manually annotated in artemis using blast and pfam searches. we could not identify a clear direct repeat indicative of a circular plasmid, as we did for psle or a putative tir characteristic of a linear plasmid, suggesting that some sequence might be missing. however, the 5’-end of psle is highly similar to the end of the tir identified for the chromosome, and includes a gene for a putative terminal helicase with 87 % and 85 % amino acid identity to sle_ and sle_ respectively. such high identity between linear chromosomes and linear plasmids is a feature of streptomyces species and suggests that psle is a linear plasmid and that this sequence is at one end of a tir. two contiguous genes were found by pfam searches to encode possible functional homologues of the partition proteins para and parb. pulse-field gel-electrophoresis of total dna isolated from s. leeuwenhoekii revealed an extrachromosomal replicon with an apparent linear size of between and  kb, consistent with the predicted size of psle as for psle we found a gene, sle2_ encoding a putative integrase, suggesting that the plasmid might be capable of integrating into the chromosome. in addition, we identified at least three genes encoding possible conjugation functions suggesting that the plasmid might be capable of self-transmission by conjugation. an interesting feature is the presence of a gene, sle2_ encoding a putative zeta-toxin e-value e-45) potentially involved in ensuring the maintenance and segregation of the plasmid; we did not find a gene encoding a putative anti-toxin, but we did find a toxin-antitoxin system encoded in the chromosome similar to that reported for s. coelicolor and streptomyces lividans. antismash did not reveal any putative natural product gene clusters, but we did find a gene cluster potentially encoding the biosynthesis of a lasso-peptide, which we have subsequently identified and characterised. intriguingly, there are several genes encoding putative transposases, many with frame shifts. this region was not present in the illumina assembly, and lies between illumina contigs and 0079; only the pacbio corrected-reads contained the full sequence. the presence of many repeated sequences with high levels of nucleotide sequence identity may explain their absence from the illumina assembly. general characteristics of the genome sequence our assembled chromosome sequence contains 7903895 bp, with a mol% g + c content of 73 %, consistent with other members of the genus streptomyces. with the proposed addition of the right hand 388 kb tir, the predicted size of the genome would be 8285171 bp. this is similar to the size of many streptomycete genomes. pcss were predicted for our assembled sequence, and pcss if we include the pcss predicted for the right tir. a table of cog functional categories was determined.table cog functional categories. cog functional categories of chromosomal protein codding sequences identified in s. leeuwenhoekii chromosome, and from s. coelicolor for comparison streptomyces leeuwenhoekii streptomyces coelicolor the s. leeuwenhoekii chromosome contains six rrna operons located between nucleotide positions and 6687647; interestingly, an orphan gene encoding a 5s rrna was found at position dnaa and dnan replication proteins are encoded in genes spanning nucleotide positions 4260071- and 4256851- respectively, indicating that the likely origin of replication of the chromosome lies between and consistent with precedent, the two rrna operons that lie between nucleotide positions and are located on the complementary strand, while the four rrna operons located between nucleotide positions and are located on the top strand. we identified trna genes, similar to the number found in other streptomyces. the general characteristics of the s. leeuwenhoekii genome are summarised in table  table general characteristics of the s. leeuwenhoekii genome biosynthetic gene clusters for specialised metabolites putative gene clusters encoding the biosynthesis of specialised metabolites were identified with antismash and non-automated searches with blast and pfam. s. leeuwenhoekii was known to produce the siderophore desferrioxamine e and the antibiotic hygromycin a; we identified probable biosynthetic gene clusters for both of these metabolites by searching for homologues of the corresponding gene clusters from s. coelicolor and streptomyces hygroscopicus nrrl respectively. the desferrioxamine e gene cluster extends from sle_ to sle_ and shows complete gene synteny with the homologous gene cluster from s. coelicolor with protein identities varying between 77 % and 93 %, while the putative hygromycin a gene cluster, which spans from sle_ to sle_ shows almost complete synteny with the homologous gene cluster from s. hygroscopicus, with protein identities of 64 % to 90 %. interestingly, antismash did not identify the hygromycin a gene cluster which lies within the proposed left tir; two putative type-i pks gene clusters and one putative hybrid type-i pks-nrps gene cluster were also found in the left tir. if, as we have proposed, the tirs span for 388 kb, then all four of these gene clusters would be duplicated at the other end of the chromosome. s. leeuwenhoekii is also known to produce two novel families of polyketide antibiotics, the chaxalactins and the ansamycin-like chaxamycins. the biosynthetic gene clusters for both antibiotics were identified and are currently under investigation. we also identified three putative gene clusters each encoding the biosynthesis of unknown lasso-peptides. two of them, the “lasso-peptide 1” and “lasso-peptide 2” gene clusters are located on the chromosome, while the “lasso-peptide 3” gene cluster is located on the linear plasmid psle all three gene clusters are currently under study in our laboratory, where pcr amplification and sanger sequencing have confirmed that the assembly and sequence reported here is correct. these clusters were not identified by antismash.  benefits and challenges of sequencing technologies: a revised pipeline to our knowledge, this is the first report of the use of ngs to produce a high quality and non-fragmented genome sequence of an actinomycete, an essential prerequisite for efficient genome mining for natural product discovery in these gc-rich bacteria. our assembly yielded the sequences of a single chromosomal contig, a complete circular plasmid, and most of a linear plasmid. although the illumina miseq assembly produced a single contig for psle it would not have been possible to confirm its circularity and completeness without the pacbio data. overall, we used two smrt cells resulting in over 120x coverage. thus, with the improving capacity of the pacbio smrt cells, we predict that a maximum of three smrt cells should be sufficient to obtain the entire complement of replicons of an actinomycete genome of around 8 mb in single contigs. this contrasts with the previously published illumina draft genome sequence of s. leeuwenhoekii that contained contigs in which we found many misassemblies and missing sequence. the pacbio long reads also permitted the identification of the start of the right hand tir, but they did not provide any sequence information for the 5 kb at the ends of the chromosome and the linear plasmid psle which was obtained from the illumina data. the reason for this is unknown, but it could reflect the different strategies used for library construction; while pacbio focuses on large fragments of ~20 kb, the average size of the illumina library used for sequencing was 550 bp. also, no specific procedure was used during dna purification to remove the protein that is covalently bound to the termini of the linear replicons, perhaps resulting in lower sequence coverage of the terminal regions that might be further exacerbated by the large fragment size used for pacbio sequencing. surprisingly, the pacbio assembly lacked the linear psle sequence, although the sequence information was present in the corrected pacbio reads. the illumina assembly contained two large contigs covering most of psle that were merged into a contiguous sequence using the pacbio corrected reads, but this plasmid would have not been identified with the pacbio assembly alone. these two findings highlight the importance of the current need to use both pacbio and illumina assemblies, instead of assembling only the pacbio data and then using the illumina reads to correct the assembly, which appears to be the accepted practice. we cannot explain the lack of assembly of some of the pacbio data present in the corrected reads but this was compensated for by using the illumina miseq assembly. in summary, for a de novo shotgun genome sequence from an actinomycete aimed at yielding single contigs per replicon, we currently propose a strategy that includes sequencing genomic dna with pacbio rsii using initially two smrt cells and a >20 kb insert library combined with illumina miseq paired-end sequencing of a 500 bp library without pcr amplification ). both data sets are assembled and compared, and the illumina contigs used to correct the pacbio nucleotide omissions/additions, which should be confirmed using gc frame plot and blast analyses. this consensus is further corrected with the illumina reads. despite the highly efficient current assembly algorithms, a considerable amount of human input was still needed to obtain a high quality single contig assembly, and accurate annotation of gene function. 
the assembled illumina sequence permitted not only the correction of omissions found in gc homopolymers in the pacbio assembly but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by pacbio. streptomyces leeuwenhoekii was isolated from the hyper-arid high-altitude atacama desert in chile and shown to produce novel polyketide antibiotics. we identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin a and desferrioxamine e, metabolites all previously shown to be produced by this strain and an additional putative gene clusters for specialised metabolites. as well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides. the s. leeuwenhoekii genome contains gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. this approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory.
 next generation dna sequencing and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. this approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. streptomyces leeuwenhoekii was isolated from the hyper-arid high-altitude atacama desert in chile and shown to produce novel polyketide antibiotics.  here we present the de novo sequencing of the s. leeuwenhoekii linear chromosome and two extrachromosomal replicons, the circular psle and the linear psle, all in single contigs, obtained by combining pacific biosciences smrt and illumina miseq technologies. we identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin a and desferrioxamine e, metabolites all previously shown to be produced by this strain and an additional putative gene clusters for specialised metabolites. as well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.  the s. leeuwenhoekii genome contains gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. this project has served to evaluate the current state of ngs for efficient and effective genome mining of high gc actinomycetes. the pacbio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. the assembled illumina sequence permitted not only the correction of omissions found in gc homopolymers in the pacbio assembly but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by pacbio. we propose an experimental pipeline that uses the illumina assembled contigs, in addition to just the reads, to complement the current limitations of the pacbio sequencing technology and assembly software. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords second/third next generation sequencingillumina miseqpacific biosciences pacbio smrtchaxamycinchaxalactinlasso peptidegenome miningissue-copyright-statement© the author 2015 