BACKGROUND
since the introduction of next generation sequencing  much attention has been given to the development of de novo assembly software.
the aim is to combine the short sequencing reads into a minimum number of linear stretches, though this goal is only partially achieved by draft assembly methods such as velvet  <cit> , soapdenovo  <cit>  or abyss  <cit> .
consequently more emphasis has been placed on so-called “genome finishing” tools which aim to reduce the number of contiguous sequences, for instance by the use of distance information between short paired reads.
indeed draft assemblies can be significantly enhanced when applying a scaffolding routine  <cit> .
nonetheless these protocols can still not overcome major hurdles such as  repeats and low-coverage regions.
alternatively the use of long read sequences as offered by the pacbio rs methodology can potentially solve complex genomic situations, yet the algorithmic implementation still suffers from a relatively high error rate.
at present so-called continuous long reads  may even exceed a size of  <dig> kbp at the cost of an error rate of approximately 15%, whereas the shorter circular consensus reads  can span maximally  <dig> kbp though at a  <dig> % error rate  <cit> .
it is a common thought that the quality of the clr reads is yet insufficient for high quality assemblies unless the genome coverage is very high.
recently, chin et al.
<cit>  have presented a novel non-hybrid method called hgap.
here only clr reads are used to finish bacterial genomes giving the advantage of a single library preparation.
although a sequence coverage of approximately 50× is sufficient to correct the error rate, a higher coverage is needed to span repeated elements.
also a significant manual intervention is needed to polish the genomes .
from a cost perspective it means that a relatively large budget is needed to close a single genome using only clr reads, especially when  eukaryotes are studied.
contemporarily it has been proposed to use a hybrid assembly approach in the attempt to enhance error-prone clr reads.
in principle this is possible using either pacbio ccs reads or short read ngs data .
to date a few algorithms have been released that are capable of upgrading pacbio clr data with high accuracy data from ccs or short read ngs data, among which pacbiotoca  <cit>  and lsc  <cit> .
these are further incorporated into hybrid assembly methods such as celera  <cit> , mira  <cit>  and allpaths-lg  <cit> .
even though promising results have been obtained, the error-correction step with short reads requires a sufficient read length  and sequencing depth, as well as large computational demands.
notably the pacbiotoca  correction pipeline also supports non-hybrid pacbio assemblies in case c <dig> or newer sequence reads are used.
as regards scaffolding, the aha  strategy is currently the most widely used approach: the method employs the usage of the clr reads only for scaffolding of pre-assembled contigs  <cit> , yet it generally results in incomplete assemblies and is not designed for large-scale genome assembly applications given the long runtime and input restrictions .
recently a novel hybrid assembly tool has been released, cerulean  <cit> , which uses abyss  <cit>  contig graph information and uncorrected clr reads to create genome scaffolds.
despite of the promising results obtained, the requirement to use abyss for the draft assembly is a limiting factor as other methods might generate a better draft  <cit> .
finally dedicated tools have been developed to close gaps within scaffolds using pacbio reads, among which pbjelly  <cit> .
a future release of this software aims to also incorporate a scaffolding module.
given the limitations encountered for short read sequencing  and pacbio long read sequencing , the complete closure of prokaryotic and eukaryotic genomes is still a relatively expensive and difficult task.
indeed it can be observed that most genome submissions consist of hundreds  of unconnected contigs.
here we present a hybrid approach to scaffold draft genomes in a cheap, fast and reliable manner.
in brief the algorithm consists of three steps: 1) alignment of long reads against the pre-assembled contigs  2) computation of contig linkage from the alignment order and 3) scaffolding of contigs  including placement of repeated elements.
on a number of test datasets we show our method, called sspace-longread, outperforms the aha method in terms of genome completeness.
importantly the input draft assemblies were constructed using only a single illumina miseq or roche  <dig> library and a complementary pacbio rs c <dig> long read library.
the combination of barcoding possibilities on the pacbio rs instrument and the relatively low coverage needed by the sspace-longread algorithm opens novel ways of scaffolding genomes at reduced costs compared for instance to the use of ngs mate paired-end libraries.
our software is setup in a user-friendly manner and is suited for analysis on small computing systems given the very fast runtime.
academics can request a free copy at http://www.baseclear.com/bioinformatics-tools/.
RESULTS
in order to test the performance of sspace-longread and compare it to pacbio’s aha scaffolds, we have performed in-depth analysis on six bacterial datasets.
these include b. trehalosi, two e. coli strains , f. tularensis, b. trehalosi, m. haemolytica and s. enterica.
details are described in table  <dig> and the methods section.
for each organism paired-end illumina miseq reads  were used to create two draft assemblies: one with ray  <cit>  and one with clcbio de novo assembly software .
an alternative draft assembly was made with newbler  using only roche  <dig> reads , except for m. haemolytica for which no  <dig> reads were available.
subsequently the draft contigs were scaffolded using uncorrected pacbio rs long clr reads  with both sspace-longread and aha.
the results are displayed in table  <dig>
overall a 90-100× illumina miseq and 45-50× roche  <dig> genome coverage is used.
for the pacbio dataset an input coverage of ~200× is available.
b. trehalosi
newbler
sspace longread
unknown
5
-
<dig> ,982
<dig> ,689
<dig>
-
-
16 min
e. coli k12
clc
sspace longread
1
1
0
<dig> ,513
<dig> ,513
<dig>
3
8
28 min
e .coli o157:h7
clc
sspace longread
10
33
2
<dig> ,369
<dig> ,184
<dig>
13
17
32 min
f. tularensis
newbler
sspace longread
3
7
0
<dig> ,474
<dig>
<dig>
16
13
14 min
m. haemolytica
clc
sspace longread
unknown
8
-
<dig> ,871
<dig> ,208
<dig>
-
-
25 min
s. enterica
newbler
sspace longread
4
4
12
<dig> ,244
<dig> ,047
<dig>
3
11
29 min
in italic-bold the platform/strategy that leads to the lowest amount of assembled scaffolds is highlighted.
the number of expected scaffolds refers to the number of chromosomes plus the number of plasmids present in the reference genome .
generally the combination 1) draft assembly using clcbio for illumina miseq reads or newbler for roche  <dig> reads and 2) scaffolding using sspace-longread for pacbio clr reads gives the best results in terms of closure and time.
notably some draft assembly contigs are not covered with pacbio reads .
the number of errors introduced during scaffolding is only limited and often are a consequence of true variations between the sequenced library and the earlier deposited reference genome.
sspace-longread effectively reconstructs  complete genomes
the assembly statistics show that both aha and sspace-longread are able to significantly reduce the amount of input draft contigs using error-prone pacbio rs clr reads as guidance.
overall the total assembly length remains relatively stable.
it is apparent that sspace-longread is better able to reconstruct continuous genome segments given the final number of scaffolds is generally lower than  <dig>  in practice this generally means a reduction of the initial amount of contigs by at least 90%.
it should also be remarked that the runtime of these tools differs by a factor of  <dig>  making our software more suited for scaffolding genomes on smaller computing systems.
in terms of accuracy  our software introduces some more errors compared to aha but this is clearly explained by the more conservative approach of aha .
also it should be remarked that some of the apparent errors are actually true variations between the sequenced strain and the reference genome, an issue which is also explained by koren et al.
<cit>  as for each sequenced strain a close  reference was selected for quality assessment.
not all input contigs were covered by pacbio reads .
this may be explained by the possible presence of short sequences such as plasmids which can not be captured by the long insert pacbio libraries.
for this reason a hybrid assembly approach provides more complete assemblies than experiments that include only one pacbio library .
another explanation can be given by presence of different dna sources in the ngs library.
for instance, in the case of s. enterica a relatively high number of sequences is generated after scaffolding the newbler draft assembly.
however  <dig> of these input contigs are not covered by pacbio reads and surprisingly most of these show a high similarity with b. taurus after a blast  <cit>  analysis on the ncbi nr database.
other contigs uncovered by pacbio show similarity with the 5386 bp genome of phi x  <dig> , which is used by illumina as a control to validate the quality of a run.
in principle the corresponding reads should be removed from the miseq runs prior to downstream analysis.
genomes become less fragmented also at a low pacbio rs clr read coverage
in figure  <dig> it can be observed that a higher pacbio rs long read coverage leads to less fragmented genomes.
nonetheless a coverage of 50× is generally sufficient to reduce the number of genome fragments to less than  <dig> scaffolds.
the best assembly results are yielded at a coverage value between 110-160×.
a higher coverage does not seem to improve the outcomes, instead this leads to more fragmented genomes.
from a cost-perspective, the pacbio xl-c <dig> chemistry specifications  imply that for a small bacteria genome closure can be achieved using one smrt cell in combination with e.g.
one miseq paired-end library.
more recent improvements of the sequencing chemistry  aim to further enhance the data throughput  and the read length .
nonetheless still a relatively high coverage is needed to completely finish a bacterial genome which is partly explained by the high error-rate which needs to be corrected for.
generally the alignment of pacbio rs long reads to a set of contigs yields low alignment scores and a non-conservative approach  will likely lead to misassemblies.
moreover it should be considered that after sequencing the number of long reads , which are essential to overcome large repeats, is relatively small compared to the number of short reads .
thus the contribution of long reads to the overall sequencing coverage is only limited.
it is to be expected though that the total data yield per smrt cell, as well as the mean read length, will significantly increase in the near future.
thus costs can be further reduced if samples are barcoded and sequenced together on the same smrt cell.
at this point a hybrid approach consisting of one short read paired-end and one long read pacbio library may eventually be the method of choice in terms of accuracy and costs compared to an alternative approach involving mate paired-end libraries.
last but not least, the computational time involved to close bacterial genomes allows high-throughput assemblies even on small compute systems.
it should be underscored that the current study was performed using uncorrected pacbio rs clr reads, thus bypassing a time-consuming error-correction step with short reads.
both the quality of the draft assembly and the selection of the assembly strategy are crucial for the success of an experiment
from table  <dig> it can be observed that the assembly results are also largely dependent on the strategy chosen for constructing the draft assembly.
common criteria used to judge a draft assembly are the number of contigs  and the n <dig> value .
nonetheless these quantitative measures do not necessarily guarantee the choice of the best assembly.it is therefore adviced to assess the quality of an assembly using a dedicated tool, such as quast  <cit> , which gives a complete overview of different quality metrics.
yet also other factors can play an important role in the selection of the most appropriate input assembly.
for example, assemblies constructed with ray do show a higher n <dig> value and a lower number of contigs compared to those constructed with clcbio or newbler.
therefore it might be intuitive to choose the ray contigs as input for sspace-longread scaffolding.
nonetheless from the sspace-longread statistics it appears to be easier to reconstruct complete genomes from clcbio or newbler contigs.
from further investigations  we noticed that assemblies constructed with ray contain repeated segments which are especially present at the contig edges.
similar observations were made for draft assemblies constructed from illumina miseq reads with spades  <cit>  and masurca  <cit>  : the amount of contigs was lower or similar to that observed for clcbio and newbler, though the errors in the draft assembly led to more  scaffolds.
although sspace-longread performs well on all tested draft assembly strategies, the user should preferably choose an assembly method that splits contigs at repeat boundaries.
consequently the assessment of the most appropriate draft assembly should not be solely based on the assembly quality-metrics, as the extent of the genome fragment reduction also depends on the draft assembly strategy.
CONCLUSIONS
we propose a novel tool for scaffolding pre-assembled contigs using long read information.
we show that even error-prone pacbio rs clr reads can be well used to connect contigs, place repeats and consequently reconstruct bacterial genomes in less than  <dig> segments when using sspace-longread.
importantly only two libraries are needed for the hybrid assembly of a bacterial genome.
one illumina miseq or roche- <dig> paired-end library is sufficient for the construction of a proper draft assembly using a state-of-the-art de bruijn-graph or overlap-consensus layout assembler, although the final quality is influenced by the exact method chosen.
importantly the number of contigs  may not be the best criteria for evaluating the best draft assembly.
indeed we show that, although draft assemblies created with ray yield fewer contigs, draft assemblies created with clcbio or newbler software lead to more closed genomes after scaffolding.
it can be therefore argued that ideally the user should choose a draft assembly method that places repeated elements into separate contigs: whereas clcbio and newbler tend to automatically break reads and contigs at repeat boundaries, ray and spades tend to merge unique and repeated sequences together in the same contigs.
we argue that one pacbio rs library is sufficient to nearly finish the bacterial assembly.
in this paper we show promising results using a pacbio coverage of 50×, a clr error-rate of 15% and a mean read length between 2-3 kb.
it is likely that further improvements of the sequencing platforms and chemistry will show additional improvements, i.e.
a complete genome closure at lower costs.
to this regard also the introduction of a barcoding system for pacbio libraries will help to use smrt cells more efficiently and enlarge the capacity per sequencing run.
also our method can be used for possible new sequencing platforms, such as the illumina moleculo and oxford nanopore systems, given that the input format  is standardized and not specifically bound to a platform.
contemporarily it can be foreseen that additional methods will appear that perform assemblies using only long read information.
at present the hgap assembly method seems to be the only such available strategy, however there are some major issues with the overlap-consensus implementation  and the introduction of erroneous duplications .
moreover our sspace-longread method is optimized for high-throughput experiments  and can be the method of choice for upgrading existing draft genomes in a cost-effective manner.
also we expect benefits for  eukaryotic organisms for which genome submissions generally consist of highly fragmented chromosomes.
we feel the current study opens new ways to address the genome assembly question and can positively contribute to the reconstruction of more complete genomic contexts.
availability and requirements
•project name: sspace-longread
•project home page:http://www.baseclear.com/bioinformatics-tools/
•operating systems: all major linux platforms
•programming languages: perl, c++
•other requirements: blasr for the alignment of long reads  <cit>
•license: basetools software license
•any restrictions to use by non-academics: commercial licence needed

 since the introduction of next generation sequencing much attention has been given to the development of de novo assembly software. the aim is to combine the short sequencing reads into a minimum number of linear stretches, though this goal is only partially achieved by draft assembly methods such as velvet, soapdenovo or abyss. consequently more emphasis has been placed on so-called “genome finishing” tools which aim to reduce the number of contiguous sequences, for instance by the use of distance information between short paired reads. indeed draft assemblies can be significantly enhanced when applying a scaffolding routine. nonetheless these protocols can still not overcome major hurdles such as repeats and low-coverage regions. alternatively the use of long read sequences as offered by the pacbio rs methodology can potentially solve complex genomic situations, yet the algorithmic implementation still suffers from a relatively high error rate. at present so-called continuous long reads may even exceed a size of kbp at the cost of an error rate of approximately 15%, whereas the shorter circular consensus reads can span maximally kbp though at a % error rate. it is a common thought that the quality of the clr reads is yet insufficient for high quality assemblies unless the genome coverage is very high. recently, chin et al. have presented a novel non-hybrid method called hgap. here only clr reads are used to finish bacterial genomes giving the advantage of a single library preparation. although a sequence coverage of approximately 50× is sufficient to correct the error rate, a higher coverage is needed to span repeated elements. also a significant manual intervention is needed to polish the genomes. from a cost perspective it means that a relatively large budget is needed to close a single genome using only clr reads, especially when eukaryotes are studied. contemporarily it has been proposed to use a hybrid assembly approach in the attempt to enhance error-prone clr reads. in principle this is possible using either pacbio ccs reads or short read ngs data. to date a few algorithms have been released that are capable of upgrading pacbio clr data with high accuracy data from ccs or short read ngs data, among which pacbiotoca and lsc. these are further incorporated into hybrid assembly methods such as celera, mira and allpaths-lg. even though promising results have been obtained, the error-correction step with short reads requires a sufficient read length and sequencing depth, as well as large computational demands. notably the pacbiotoca correction pipeline also supports non-hybrid pacbio assemblies in case c or newer sequence reads are used. as regards scaffolding, the aha strategy is currently the most widely used approach: the method employs the usage of the clr reads only for scaffolding of pre-assembled contigs, yet it generally results in incomplete assemblies and is not designed for large-scale genome assembly applications given the long runtime and input restrictions. recently a novel hybrid assembly tool has been released, cerulean, which uses abyss contig graph information and uncorrected clr reads to create genome scaffolds. despite of the promising results obtained, the requirement to use abyss for the draft assembly is a limiting factor as other methods might generate a better draft. finally dedicated tools have been developed to close gaps within scaffolds using pacbio reads, among which pbjelly. a future release of this software aims to also incorporate a scaffolding module. given the limitations encountered for short read sequencing and pacbio long read sequencing, the complete closure of prokaryotic and eukaryotic genomes is still a relatively expensive and difficult task. indeed it can be observed that most genome submissions consist of hundreds of unconnected contigs. here we present a hybrid approach to scaffold draft genomes in a cheap, fast and reliable manner. in brief the algorithm consists of three steps: 1) alignment of long reads against the pre-assembled contigs 2) computation of contig linkage from the alignment order and 3) scaffolding of contigs including placement of repeated elements. on a number of test datasets we show our method, called sspace-longread, outperforms the aha method in terms of genome completeness. importantly the input draft assemblies were constructed using only a single illumina miseq or roche library and a complementary pacbio rs c long read library. the combination of barcoding possibilities on the pacbio rs instrument and the relatively low coverage needed by the sspace-longread algorithm opens novel ways of scaffolding genomes at reduced costs compared for instance to the use of ngs mate paired-end libraries. our software is setup in a user-friendly manner and is suited for analysis on small computing systems given the very fast runtime. academics can request a free copy at http://www.baseclear.com/bioinformatics-tools/.  in order to test the performance of sspace-longread and compare it to pacbio’s aha scaffolds, we have performed in-depth analysis on six bacterial datasets. these include b. trehalosi, two e. coli strains, f. tularensis, b. trehalosi, m. haemolytica and s. enterica. details are described in table  and the methods section. for each organism paired-end illumina miseq reads were used to create two draft assemblies: one with ray and one with clcbio de novo assembly software. an alternative draft assembly was made with newbler using only roche reads, except for m. haemolytica for which no reads were available. subsequently the draft contigs were scaffolded using uncorrected pacbio rs long clr reads with both sspace-longread and aha. the results are displayed in table  overall a 90-100× illumina miseq and 45-50× roche genome coverage is used. for the pacbio dataset an input coverage of ~200× is available. b. trehalosi newbler sspace longread unknown 5 -,982,689 - - 16 min e. coli k12 clc sspace longread 1 1 0,513,513 3 8 28 min e.coli o157:h7 clc sspace longread 10 33 2,369,184 13 17 32 min f. tularensis newbler sspace longread 3 7 0,474 16 13 14 min m. haemolytica clc sspace longread unknown 8 -,871,208 - - 25 min s. enterica newbler sspace longread 4 4 12,244,047 3 11 29 min in italic-bold the platform/strategy that leads to the lowest amount of assembled scaffolds is highlighted. the number of expected scaffolds refers to the number of chromosomes plus the number of plasmids present in the reference genome. generally the combination 1) draft assembly using clcbio for illumina miseq reads or newbler for roche reads and 2) scaffolding using sspace-longread for pacbio clr reads gives the best results in terms of closure and time. notably some draft assembly contigs are not covered with pacbio reads. the number of errors introduced during scaffolding is only limited and often are a consequence of true variations between the sequenced library and the earlier deposited reference genome. sspace-longread effectively reconstructs complete genomes the assembly statistics show that both aha and sspace-longread are able to significantly reduce the amount of input draft contigs using error-prone pacbio rs clr reads as guidance. overall the total assembly length remains relatively stable. it is apparent that sspace-longread is better able to reconstruct continuous genome segments given the final number of scaffolds is generally lower than in practice this generally means a reduction of the initial amount of contigs by at least 90%. it should also be remarked that the runtime of these tools differs by a factor of making our software more suited for scaffolding genomes on smaller computing systems. in terms of accuracy our software introduces some more errors compared to aha but this is clearly explained by the more conservative approach of aha. also it should be remarked that some of the apparent errors are actually true variations between the sequenced strain and the reference genome, an issue which is also explained by koren et al. as for each sequenced strain a close reference was selected for quality assessment. not all input contigs were covered by pacbio reads. this may be explained by the possible presence of short sequences such as plasmids which can not be captured by the long insert pacbio libraries. for this reason a hybrid assembly approach provides more complete assemblies than experiments that include only one pacbio library. another explanation can be given by presence of different dna sources in the ngs library. for instance, in the case of s. enterica a relatively high number of sequences is generated after scaffolding the newbler draft assembly. however of these input contigs are not covered by pacbio reads and surprisingly most of these show a high similarity with b. taurus after a blast analysis on the ncbi nr database. other contigs uncovered by pacbio show similarity with the 5386 bp genome of phi x, which is used by illumina as a control to validate the quality of a run. in principle the corresponding reads should be removed from the miseq runs prior to downstream analysis. genomes become less fragmented also at a low pacbio rs clr read coverage in figure  it can be observed that a higher pacbio rs long read coverage leads to less fragmented genomes. nonetheless a coverage of 50× is generally sufficient to reduce the number of genome fragments to less than scaffolds. the best assembly results are yielded at a coverage value between 110-160×. a higher coverage does not seem to improve the outcomes, instead this leads to more fragmented genomes. from a cost-perspective, the pacbio xl-c chemistry specifications imply that for a small bacteria genome closure can be achieved using one smrt cell in combination with e.g. one miseq paired-end library. more recent improvements of the sequencing chemistry aim to further enhance the data throughput and the read length. nonetheless still a relatively high coverage is needed to completely finish a bacterial genome which is partly explained by the high error-rate which needs to be corrected for. generally the alignment of pacbio rs long reads to a set of contigs yields low alignment scores and a non-conservative approach will likely lead to misassemblies. moreover it should be considered that after sequencing the number of long reads, which are essential to overcome large repeats, is relatively small compared to the number of short reads. thus the contribution of long reads to the overall sequencing coverage is only limited. it is to be expected though that the total data yield per smrt cell, as well as the mean read length, will significantly increase in the near future. thus costs can be further reduced if samples are barcoded and sequenced together on the same smrt cell. at this point a hybrid approach consisting of one short read paired-end and one long read pacbio library may eventually be the method of choice in terms of accuracy and costs compared to an alternative approach involving mate paired-end libraries. last but not least, the computational time involved to close bacterial genomes allows high-throughput assemblies even on small compute systems. it should be underscored that the current study was performed using uncorrected pacbio rs clr reads, thus bypassing a time-consuming error-correction step with short reads. both the quality of the draft assembly and the selection of the assembly strategy are crucial for the success of an experiment from table  it can be observed that the assembly results are also largely dependent on the strategy chosen for constructing the draft assembly. common criteria used to judge a draft assembly are the number of contigs and the n value. nonetheless these quantitative measures do not necessarily guarantee the choice of the best assembly.it is therefore adviced to assess the quality of an assembly using a dedicated tool, such as quast, which gives a complete overview of different quality metrics. yet also other factors can play an important role in the selection of the most appropriate input assembly. for example, assemblies constructed with ray do show a higher n value and a lower number of contigs compared to those constructed with clcbio or newbler. therefore it might be intuitive to choose the ray contigs as input for sspace-longread scaffolding. nonetheless from the sspace-longread statistics it appears to be easier to reconstruct complete genomes from clcbio or newbler contigs. from further investigations we noticed that assemblies constructed with ray contain repeated segments which are especially present at the contig edges. similar observations were made for draft assemblies constructed from illumina miseq reads with spades and masurca: the amount of contigs was lower or similar to that observed for clcbio and newbler, though the errors in the draft assembly led to more scaffolds. although sspace-longread performs well on all tested draft assembly strategies, the user should preferably choose an assembly method that splits contigs at repeat boundaries. consequently the assessment of the most appropriate draft assembly should not be solely based on the assembly quality-metrics, as the extent of the genome fragment reduction also depends on the draft assembly strategy.  we propose a novel tool for scaffolding pre-assembled contigs using long read information. we show that even error-prone pacbio rs clr reads can be well used to connect contigs, place repeats and consequently reconstruct bacterial genomes in less than segments when using sspace-longread. importantly only two libraries are needed for the hybrid assembly of a bacterial genome. one illumina miseq or roche- paired-end library is sufficient for the construction of a proper draft assembly using a state-of-the-art de bruijn-graph or overlap-consensus layout assembler, although the final quality is influenced by the exact method chosen. importantly the number of contigs may not be the best criteria for evaluating the best draft assembly. indeed we show that, although draft assemblies created with ray yield fewer contigs, draft assemblies created with clcbio or newbler software lead to more closed genomes after scaffolding. it can be therefore argued that ideally the user should choose a draft assembly method that places repeated elements into separate contigs: whereas clcbio and newbler tend to automatically break reads and contigs at repeat boundaries, ray and spades tend to merge unique and repeated sequences together in the same contigs. we argue that one pacbio rs library is sufficient to nearly finish the bacterial assembly. in this paper we show promising results using a pacbio coverage of 50×, a clr error-rate of 15% and a mean read length between 2-3 kb. it is likely that further improvements of the sequencing platforms and chemistry will show additional improvements, i.e. a complete genome closure at lower costs. to this regard also the introduction of a barcoding system for pacbio libraries will help to use smrt cells more efficiently and enlarge the capacity per sequencing run. also our method can be used for possible new sequencing platforms, such as the illumina moleculo and oxford nanopore systems, given that the input format is standardized and not specifically bound to a platform. contemporarily it can be foreseen that additional methods will appear that perform assemblies using only long read information. at present the hgap assembly method seems to be the only such available strategy, however there are some major issues with the overlap-consensus implementation and the introduction of erroneous duplications. moreover our sspace-longread method is optimized for high-throughput experiments and can be the method of choice for upgrading existing draft genomes in a cost-effective manner. also we expect benefits for eukaryotic organisms for which genome submissions generally consist of highly fragmented chromosomes. we feel the current study opens new ways to address the genome assembly question and can positively contribute to the reconstruction of more complete genomic contexts. availability and requirements •project name: sspace-longread •project home page:http://www.baseclear.com/bioinformatics-tools/ •operating systems: all major linux platforms •programming languages: perl, c++ •other requirements: blasr for the alignment of long reads •license: basetools software license •any restrictions to use by non-academics: commercial licence needed 
on a test set comprising six bacterial draft genomes, assembled using either a single illumina miseq or roche library, we show that even a 50× coverage of uncorrected pacbio rs long reads is sufficient to drastically reduce the number of contigs. the recent introduction of the pacific biosciences rs single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. we conclude that the recent advances of the pacbio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using pacbio rs long read information as a backbone.
 the recent introduction of the pacific biosciences rs single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. the long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from next generation sequencing data.  here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using pacbio rs long read information as a backbone. on a test set comprising six bacterial draft genomes, assembled using either a single illumina miseq or roche library, we show that even a 50× coverage of uncorrected pacbio rs long reads is sufficient to drastically reduce the number of contigs. comparisons to the aha scaffolder indicate our strategy is better capable of producing complete bacterial genomes.  the current work describes our sspace-longread software which is designed to upgrade incomplete draft genomes using single molecule sequences. we conclude that the recent advances of the pacbio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. de novo assemblyscaffoldingsingle molecule sequencingpacific biosciencesgenome finishing 