Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products

19 Pages·2016·1.44 MB·English

marine drugs
Review
Next Generation Sequencing of Actinobacteria for the
Discovery of Novel Natural Products
JuanPabloGomez-Escribano*,SilkeAltandMervynJ.Bibb
DepartmentofMolecularMicrobiology,JohnInnesCentre,NorwichResearchPark,Norwich,NR47UH,UK;
[email protected](S.A.);[email protected](M.J.B.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:PaulLong
Received:25February2016;Accepted:6April2016;Published:13April2016
Abstract: Like many fields of the biosciences, actinomycete natural products research has been
revolutionisedbynext-generationDNAsequencing(NGS).Hundredsofnewgenomesequencesfrom
actinobacteriaaremadepubliceveryyear,manyofthemasaresultofprojectsaimedatidentifying
newnaturalproductsandtheirbiosyntheticpathwaysthroughgenomemining. Advancesinthese
technologies in the last five years have meant not only a reduction in the cost of whole genome
sequencing,butalsoasubstantialincreaseinthequalityofthedata,havingmovedfromobtaininga
draftgenomesequencecomprisedofseveralhundredshortcontigs,sometimesofdoubtfulreliability,
tothepossibilityofobtaininganalmostcompleteandaccuratechromosomesequenceinasingle
contig,allowingadetailedstudyofgeneclustersandthedesignofstrategiesforrefactoringandfull
geneclustersynthesis. Theimpactthatthesetechnologiesarehavinginthediscoveryandstudyof
naturalproductsfromactinobacteria,includingthosefromthemarineenvironment,isonlystarting
toberealised. Inthisreviewweprovideahistoricalperspectiveofthefield,analysethestrengths
andlimitationsofthemostrelevanttechnologies,andsharetheinsightsacquiredduringourgenome
miningprojects.
Keywords: actinomycetes;Streptomyces;genomemining;PacBio;Illumina;nextgenerationsequencing
1. Introduction
Actinobacteriaproducemorethan70%ofallnaturalproductscaffoldsusedforthemanufactureof
clinically-relevantanti-infectives[1].Duringthefinaldecadesofthelastcentury,effortstodiscovernew
anti-infectivesofmicrobialoriginwerealmostabandoned;however,thebeginningofthiscenturyhas
seenareturntothesearchfornovelbioactivenaturalproductsfromactinobacteria,duetoadvances
in cultivation and activity screening [2,3] and, in particular for this review, the realisation of the
biosyntheticpotentialofactinobacteria,encodedinthegenomebutnotexpressedascompounds,and
thedevelopmentofgeneticapproachestoaccessthishiddenpotential[3,4].
DuringthesequencingoftheStreptomycescoelicolorgenomeinthelate1990s[5]itbecameevident
that actinomycetes carry the genetic potential to produce many more natural products than those
detected under laboratory conditions, and during the following years many previously-unknown
metabolitesproducedbyS.coelicolorwereidentifiedandcharacterised[6]. Itwasrealisedthenthat
accesstothegenomesequenceofastraincouldbeusedtounlockthebiosyntheticpotentialofthe
micro-organism using different molecular genetic approaches [1,6–8] in a strategy that has been
called “genome mining” [7]. However, in the early 2000s the only automated technique for DNA
sequencingwasthedideoxynucleotidemethoddevelopedbySangerandco-workers[9];evenwith
thedevelopmentsreachedinthe2000s,includingthoseincomputingandassemblyalgorithms[10],
Sangersequencingwastooexpensiveandlabourintensivetoprovidesufficientcoverageforaroutine
Mar.Drugs2016,14,78;doi:10.3390/md14040078 www.mdpi.com/journal/marinedrugs
Mar.Drugs2016,14, 78 2of19
whole-genomeshotgunapproach,requiringthecreation,sorting,andsequencingofgenomiclibraries
priortofullgenomeassembly[5,10].
Sincethelate2000swehaveseenthecontinuousreleaseofnewDNAsequencingtechnologies
thathavepushedforwardbothsequencingcapacityandaccuracy,andloweredthecostpersequenced
nucleotide. Thesetechnologiesarereferredtoasnext-generationsequencing(NGS)andaredefinedas
“non-Sanger-based,high-throughput,andeliminatingtheneedforfragment-cloningandamplification
inEscherichiacolipriortosequencing”(adaptedfrom[11]). NGStechnologiesmakeaffordablethe
high-throughputsequencingofbacterialgenomeswhich,whencoupledwithacontinuousadvance
incomputingalgorithmsanddatabasesfortheautomatedscanningandannotationofspecialised
metabolitegeneclusterslikeAntiSMASH[12]andMIBiG[13],areenablingthecontinuousdiscovery
andstudyofnaturalproductsbiosyntheticpathwaysbygenomemining.
The relatively recent realisation that actinobacteria from our oceans and seas are much more
abundantthanpreviouslythoughtprovidesanewopportunityforthediscoveryofdrugsfromthe
marine environment. Coupled with the difficulty of growing some of these organisms at a large
scale,thesequence-basedapproachtonaturalproductdiscoverydescribedhereshouldbeparticularly
pertinentanduseful.
2. AShortWalkthroughNGSTechnologies
ThefirstNGStechnologiestoappear,referredtoassecond-generationsequencing(SGS),reliedon
cyclesoftheterminationofDNApolymerisationandrecordingoftheincorporatednucleotidesineach
cycle.ThefirstSGStechnologytobecommercialised,inmid-2005,was454pyrosequencing(by454Life
Sciences,nowasubsidiaryofRocheDiagnostics)[14],followedin2006bythereversible-terminator
chemistryofSolexa/Illumina(bySolexaLtd.,nowIlluminaInc.)[15]which,becauseofitslowercost,
highthroughput,andaccuracy[16],hasbecomethefirstchoicesequencingtechnologyacrossmany
fieldsofresearchandmedicaldiagnostics. OtherSGStechnologieshavebeencommercialisedbuthave
notattainedthepopularityofthesetwo. Examplesthathavebeenusedforactinobacteriagenome
sequencingareSOLiD(releasedin2006byAppliedBiosystemsInc.,nowThermoFisherScientific/Life
Technologies) and Ion Torrent (released in 2010 by Ion Torrent Systems Inc., now Thermo Fisher
Scientific/LifeTechnologies).
Theyear2011sawthecommercialisationofthefirstthird-generationsequencing(TGS)technology:
SingleMoleculeRealTime(SMRT)(byPacificBiosciencesofCalifornia,Inc.) usuallyreferredtoas
“PacBio” [17]. Another TGS technology currently in development, but already very promising, is
Nanopore DNA sequencing (by Oxford Nanopore Technologies Ltd.); this platform is accessible
throughanearlyaccessprogram[18];earlyreportsindicatethatthetechnology,despitereadaccuracy
under 70%, is already useful for scaffolding, thanks to read lengths of over 1 kb and up to 98 kb;
however,theapplicationtoactinobacterialgenomesequencingisstillheavilyhamperedbythehigh
mol%G+Coftheorganisms[19,20].
TGStechnologies,asopposedtoSGS,relyonsequencingsinglemoleculeswithoutamplification
(which can create problems with even genome representation) and in real-time (no cycles of
polymerisation/termination) and are capable of providing read lengths of several kilobases [21]
facilitatingtheassemblyofwhole-genomeshotgunprojects.
3. ChallengesofActinobacterialGenomics
Thehighmol%G+Ccontentofactinomycetegenomesposesdifficultiesnotjustforthesequencing
technologyitselfbutalsotothecomputingalgorithmsusedfortheassembly[22],althoughmanyof
theerrorsandsequencingbiasesduetomol%G+Cbiashavebeenlessenedbyimprovementsinlibrary
preparation[23,24]. Amorespecificproblemispresentedbythelinearchromosomeandplasmids
ofmanyimportantactinobacteria,likethestreptomycetes,withlongterminalinvertedrepeatsthat
canreachoveronemegabase[25], impossibletoresolvewithcurrentsequencingtechnologies. In
addition, extraction of high molecular weight DNA of the high quality required for NGS library
Mar.Drugs2016,14, 78 3of19
construction,andinparticularofTGS,isnottrivialandinmanycasescurrentlynotfeasible,especially
fromactinobacteriadifficulttocultureandresilienttocellwalldigestion.
ManyofthemostrelevantnaturalproductsbelongtothechemicalfamiliesofTypeIpolyketides
andnon-ribosomalpeptides[26]. Thebackboneofthesecompoundsissynthesisedbylargeenzymes,
polyketide synthases (PKS), and non-ribosomal peptide synthetases (NRPS), which consist of a
highly-conservedmodularenzymaticarchitecturethatisreflectedatthenucleotidesequencelevel
byhighlysimilarintragenicandintergenictandemrepeats, frequentlyspanningover700bp; e.g.,
S. coelicolor coelimycin PKS gene sco6274 positions 3879–4533 and sco6275 11986–12639 share 99%
intergenic identity (Figure 1); and the calcium-dependent antibiotic NRPS gene sco3230, positions
13187–14121and16307–17241,share95%intragenicidentity. Theserepeatsare,inmanycases,longer
thantheread-lengthofallSGStechnologies,makingitverydifficult,ifnotimpossible,tocorrectly
assembletheseveryimportantgeneclusters.
4. TheRead-LengthProblem
Whenwetalkabout“assembly”wemeanthedeterminationofthecorrectorderofthereads,the
thousandsofshortoverlappingfragmentsinwhichthewholegenomesequencewouldberepresented
(see [27] for a good review). Due to simple statistics, the longer and more accurate the reads are,
thelongerandmorespecifictheoverlapbetweentworeadswillbeand,therefore,amorereliable
assemblycanbecomputed[28].
OftheSGStechnologies,454hasprovided,consistently,thelongestreadlengthanddominated
actinobacterial genome sequencing until 2012 when Illumina took over with an increasing
high-accuracy read-length, together with the highest output and lowest cost per base. Currently,
withmaximumpaired-end(i.e.,bothendsofthesameDNAmoleculearesequenced)readlengths
of around 2 ˆ 150 nt for Illumina’s highest throughput HiSeq sequencers, and around 2 ˆ 300 nt
forthelowerthroughputMiSeq(source: www.Illumina.com; accessedonOctober2015), Illumina
technologycantypicallydeliveramaximumof500ntofcontiguousreliablesequence,insufficientto
resolvethehighlyrepetitivePKSandNRPSgenes,ribosomalRNAoperons(whichspanabout5.5kb),
and terminal inverted repeats. Of the other SGS technologies, only 454-pyrosequencing provides
longerreadlengths,700ntandupto1kbaccordingtothemanufacturer(GSFLX+instrument;source:
http://454.com;accessedonOctober2015). IonTorrentiscurrentlyofferingupto400ntreadsand
presentsanalternativetoIlluminafordenovosequencingofsmallgenomes[29].
ThissituationhaschangeddramaticallywiththeadventofTGStechnologies. PacBioSMRT,the
onlyTGStechnologycurrentlycommercialised,isstilluniqueinitsabilitytoresolvehighly-similar
repetitive sequences thanks to an average read-length of over 10 kb [30] at an affordable 100 fold
coverage required, not just for a reliable assembly, but also for an accurate base-calling of the
consensussequence.
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Mar. Drugs 2016, 14, x 4 of 20
A

B

79 % identity

C
p) p)
b b
O6275 (13674 O6274 (10731
C C
S S
SCO6275 (13674 bp) SCO6275 (13674 bp)

D

Figure1. Exampletoillustratetheproblemofintergenicandintragenicsequenceidentityingenes
encoding modular polyketide synthases. (A) Screenshot from Artemis software [31] showing the

geneticorganisationofthefirsttwoPKSgenesofthecoelimycingenecluster;thehighlightedsegments
correspondtothelastmoduleofSco6275andfirstofSco6274(B)whichshareasequenceidentityof
79%over5kb(grey)andmorethan99.5%identityover650bp(orange).Acloserlookatthenucleotide
sequenceofthesegenesbyBLAST(dotplotsandalignmentsgeneratedatNCBIserver)showsthe
intragenic(C,left)andintergenic(C,right)regionsofhighidentity,andthenucleotidealignmentofthe
regioningrey(D,left)andorange(D,right,highlightedinblue).KS,ketosynthase;AT,acyltransferase;
DH,dehydratase;KR,ketoreductase;ACP,acyl-carrierprotein.
Mar.Drugs2016,14, 78 5of19
5. HistoricalPerspectiveofActinobacterialGenomeSequencing
The first actinobacterial genome fully sequenced was that of Mycobacterium tuberculosis [32],
and the first genome of an actinobacterium relevant for natural products was that of the model
streptomyceteS.coelicolor[5],followedbytheindustriallyimportantStreptomycesavermitilis[33],both
usingSangersequencing. Asstandardlypracticedatthetime,theS.coelicolorgenomewasassembled
bysequencinganorderedcosmidandBACgenomiclibrary. Incontrast,sequencingofthegenome
ofS.avermitiliswasfirstattemptedusingawhole-genomeshotgunapproach;thepublishedarticle
providesenoughdetailforustograspthedifficultiesofawhole-genomeshotgunapproachonlya
decadeago;twogenomecontigsof9,025,608ntand94,287nt(thechromosomeandalinearplasmid,
respectively;atotalof9,119,895nt)wereassembledinitiallyfrom186,619sequencesfromrandom
~2kbfragments(estimating600ntofhigh-qualitybase-calls,(186,619ˆ600)nt/9,119,895nt=12.3
foldcoverage);thisdatawasnotsufficientforagoodqualityassemblyandtheauthorshadtorelyon
theend-sequencingofmorethan10,000cosmidsfromagenomiclibrary,overahundredfullcosmid
inserts, and162PCR-productsequences, altogetherreaching13.3foldcoveragewithdirectedgap
filling[33]. ThisexampledemonstratesthatshotgunsequencingwithexclusiveuseofSanger-based
techniqueswasextremelylaboriousandnotsufficientforefficientfullgenomeshotgunassembly.
TheSGStechnologiesdidallowshotgunsequencingofwholegenomesatmuchhighercoverage,
buttheshortread-lengthcombinedwiththeintrinsicdifficultiesofactinobacterialgenomeshampered
theassemblyoffullrepliconsinsinglecontigs. Duetothelongerread-length,454-pyrosequencing
hasbeenthetechnologyofchoiceforachievingamorecompletegenomeassembly,butfullreplicon
assemblyintoasinglecontighasrequiredtheuseofSangerend-sequencingoflarge-insertgenomic
librariesanddirectedgap-fillingbyPCR-amplification. Examplesarethesequencingofthegenomes
ofStreptomyceshygroscopicus5008, inwhichIlluminadatawasusedtocorrectbase-callerrors[34],
andofStreptomycesalbusJ1074forwhich,evenwitha377foldcoveragefromcombinedIllumina-454
sequencingdataofthreelibrariesofdifferentinsert-size,alargeBAClibrarywasend-sequencedand
chromosome-walkingusedtofillgapsandmergetheinitial76contigs[35].
6. TheExplosionofGenomeMining
Despitethedifficultyincompletinganaccuratefullgenomesequenceofanactinobacterium,the
numberofavailablegenomeassemblies(ofdifferentcompletenessandqualitylevel)inthedatabases
hasgrownexponentiallyduringthepastdecade(Figure2). Themainreasonisthatacompleteand
accurategenomesequenceisnotaprerequisitefortheapplicationofthegenomeminingapproach
to natural product discovery; multi-contig genome drafts can be equally useful when it comes to
identifyingthegeneclusterthatdrivesthesynthesisofaknowncompound,orgeneclustersforthe
biosynthesisofunknownbutpotentiallyinterestingnaturalproducts. Themostcommonautomated
pipelineforsearchingandannotatingnaturalproductsbiosyntheticgeneclusters,AntiSMASH[12],
caneasilydealwithmulti-contiggenomeassemblies.
EvenintheearlydaysofSolexatechnology,theinformationobtainedcouldsufficefordesigning
strategiesforgenomemining. Fortheidentificationandcloningofthemicrobisporicinbiosynthetic
genecluster,almost900MbofsequencedatafortheMicrobisporacorallinagenomewasobtained,but
despitethehighcoverage(greaterthan100ˆ)theshortreadlengthatthetime,only36nt,allowed
onlyaveryfragmentedassembly(seeTable1). However,thisprovidedenoughinformationtoidentify
putativehomologsoflantibioticbiosyntheticgenes,todesignprobesforSouthernBlotting,andto
successfullyscreenacosmidlibrarytoobtaintheentiregenecluster[36,37]. Genomesequencingwith
Solexawasalsocrucialintheidentificationandcloningofthecypemycingeneclusterafterstrategies
basedonhybridisationscreeningofacosmidlibrarywereunsuccessful[38].
Atthesametime,454pyrosequencing,withitslongerreads(200–400ntatthetime),provided
aclearadvantageforactinobacterialgenomeassembly;sequencingofM.corallinagenomewithone
quarterofarunof454yieldedmuchlessdatathanSolexa,butledtoanassemblywithfewerand
longer contigs that allowed the identification of more putative lantibiotic biosynthetic genes and,
Mar.Drugs2016,14, 78 6of19
crucially,thegeneencodingtheputativeprecursorpeptidebyusingtheaminoacidsequenceasa
queryMafro. DrraugtsB 2L01A6, S14T, xn search[37]. Theplanosporicin[39,40],tunicamycin[41],andbottrom8 oyfc 2i0n [42]
biosyntheticgeneclusterswerealsoclonedonthebasisofdraftgenomesequencesobtainedwith454
in this review; while many of these actinobacteria might be relevant to natural products research, the
(seeTable1forasummaryofourlab’sexperience).
low number of actual species filtered out is not likely to influence the conclusions of this review.

FigurFeig2u.rNe u2.m Nbuermobfegr eonf ogmeneosm(leesf t)(laenftd) aansdse masbselimesbl(ireisg h(rti)gohft)a octf inaoctbinaocbtearcitaersipa escpieescieres lerevlaenvatntot ntoa tural
prodnuacttusrrael speraordcuhcdtse rpeosesaitrecdh daetpNoCsitBeIdd aat tNabCaBsIe dsaptaebrayseesa pr.er year.
PATRIC allows a more comprehensive perspective on the use of technologies than NCBI
Table1. Summaryofourgroup’sexperienceusingNGSforgenomemining. OnlytheStreptomyces
(which seems not to contain searchable information prior to 2010); however, it is still not easy to
leeuwenhoekii genome was published in full; for the others, only the relevant and confidently
analyse the information (the names used vary, e.g., 454 can be referred to with just “GS FLX”) and
re-sequencedsegmentswerepublished.
make any search and sorting very difficult, and there were 118 genomes sequenced with more than
one technology, almost always involving 454 andN I5ll0uminaL,o anngeds tthere were over 300 entries without
Numberof Totalsum Lanes/
Micarnoo ragsasnoiscmiatedTe cthencohlnogoylogy)Y eaanrd, consceoqntuigesn1tly, wceo nctihgose toc odntoigwnloaodf cothntei gAs ctinTootbaladcattearia gruennsomesR ef.
(nt) (nt)
Mtiacrbobleis paonrad analyse it manually. The picture painted by our analysis (Figure 3) shows that Illumina is
Solexa/
bcoyr alflainra the mIollsutm icnoammoMnildy2 0u07sed tec1h4n39o5logy, du1e6 3to the 4lo43w6 cost p2.e93r Mnbucleot8id81eM, bthe ver7y high[3 7]
NRRL30420
Mtihcrroobiuspgorhaput, and the wide availability of suppliers. As mentioned above, the longer reads of 454
pcoyrarlolisneaquencing45 4made tMhiids 2t0e0c8hnol7o5g80y(,3 0d27e)spite 1i2ts19 higher 89c1o3st, the 4.c6h4oMibce whe2n8 Mab more1 /p4recise[3 7]
NRRL30420
Starespsteommycbelsy wasS odlexeas/ired, in particular for PKS and NRPS gene clusters, and so it was the main
Mid2007 15,471 378 7830 8.5Mb [38]
spt.eOcHhn-4o15lo6gy usIleludm uinnatil just two years ago, when Illumina took off. A continuous presence of Sanger
Planomonospora
seaqlbuaencing refl4e5c4ts the uMseid o2f0 0th9is te3c0h6n6o(1l6o1g8)y to co7m56plemen1t4 ,t7h67e limita2.t3i2oMnsb of NG1S3 Mwbhen th1e/ g8oal is[4 0]
NtRoR Lo1b8t9a2i4n a finished genome; end-sequencing of genomic library clones and sequencing of PCR
Planomonospora
amalpbalicons to f4i5ll4 gaps Minid 2t0h1e1 asse1m01b7l(y94 4a)re th1e7, 3m14ost c1o4m1,1m00on ap7p.3liMcabtions o72f MSbanger.1 /S4OLiD[4 0]
NtReRcLh1n8o92lo4gy has been a very minor player with only 2–4 entries in PATRIC and NCBI, respectively.
Streptomyces
cIhnarttereruessistingly, Io45n4 Torrent2 0s0e8ems to h3a1v12e gained4 5m82oment5u3m,91 6in the7 .p95aMstb year; 28s6inMcbe mos1t/ 2of the[4 1]
NsRuRbLm38i8tt2ed projects originated at the same institution, this may not reflect wider adoption, although
Streptomyces
boimttroppreonsvisements 4in54 libraryE cnodn20s1tr0uction46 3w(i4t2h7) high m40,o44l%0 G+C1 8D3,4N03A [49]8 a.8n5dM bbioinfo1r1m5Mabtics [501/] 4might[4 2]
DhSeMlp40 t2h62is technology to be more widely embraced in the future.
Streptomyces Full
leeuwenhAoeskisiemblIilMelusim SoeinqbataineMd iuds2i0n1g3 TGS(13 7P857as(cc2Ba7f9i.o))2 were5 9s,2u8b4mitte1d5 7t,o22 t5he dat8a.1bMasbes from(17.12 2250MG1bb2), but( 5t0h0e first[4 3]
DeSxMam421p2l2es made use primarily of Illumina and 454 SGS, using limited coverage obtained witchyc Pleas)cBio
Streptomyces
leefuowre nshcoaefkfiioldingP aocfB icoontigsE nadn2d0 1g3ap closin3g [51,52]7., 895,833 7,895,833 8Mb 966Mb 2(3) [43]
cells
DSM42122
1Totalnumberofcontigsisgivenfirst;whenavailable,thenumberofcontigslargerthan500ntisgivenin
brackets.2Numberofscaffoldsinwhichcontigswerejoined.
Partialgenomesequenceinformationhasalsobeenusedtodesignsuccessfulstrategiestoobtain
or identify the unknown metabolic product of a biosynthetic gene cluster. Three main strategies
are pursued: the activation of the expression of the gene cluster in the natural producer, cloning
andexpressioninaheterologoushost,andmutationfollowedbymetaboliteprofiling. Anelegant
exampleofthefirstapproachistheidentificationandcharacterisationofstambomycin;Lauretiand
co-workersfoundacrypticgenecluster(i.e.,withoutaknownproduct)encodingthebiosynthesisof
anunknownpolyketideinthedraftgenomesequenceofStreptomycesambofaciensand,afterinferring
Mar.Drugs2016,14, 78 7of19
the transcriptional regulatory network, they induced the production of the metabolic product by
constitutive expression of a cluster-situated gene encoding a transcriptional activator of the LAL
family [44]. A similar example is the discovery and characterisation of ansamycin compounds
with novel chemistry from Streptomyces sp. LZ35 [45]. An example of the second approach is the
characterisationofgrisemycin,alinaridinfromStreptomycesgriseusIFO13,350,whichwaspurified
afterheterologousexpressionoftheclonedgenecluster[46]. Thethirdapproachcanbeusedwhen
thegeneclusterisexpressedunderlaboratorycultureconditions,butitsproductisnotknown: the
genomesequencecanbeusedtodesigngeneknock-outsthat,togetherwithcomparativemetabolite
profilingoftheproducingandnon-producingstrains,allowstheidentificationofthemetabolicproduct
(e.g.,[47]).
Thus, despite the large number of contigs, and most likely many misassembly issues, draft
genome sequences can, indeed, be sufficient to successfully identify new natural products or the
biosyntheticgeneclustersofknowncompounds. Dozensofresearchgroupsacrosstheglobehave
adoptedoneormoreoftheseapproaches,withmorethan120papersonthistopicpublishedbythe
endof2015(numberofrecordsfoundinPubMedwiththesearchstring“(“genomemining”)AND
(streptomycesORactinobacteriaORactinomycetesORstreptomycetes)”)ofwhich70wereinthelast
twoyears(notethatsincePubMeddoesnotindexmanyjournalsrelevanttothefield,thisislikelyto
beanunderestimateofthenumberofrelevantpublications). Therefore,itshouldnotbesurprising
thatthenucleotidesequencedatabasescontainover2000genomeassembliesofactinobacterialspecies.
Although the situation is improving with more stringent and precise requirements during
sequence submission, it is still difficult to obtain accurate characteristics about genome sequences
availableinpublicdatabases. Thereisalsoconfusionabouttheterminologyusedbyeachdatabase;
e.g.,NCBIhostsadatabasenamed“Assembly”thatcontainsreleasedsequencesatdifferentstagesof
completion;entriesinthe“Genome”databasecontain“assemblies”groupedatthespecieslevel,even
withdifferentstrainsinthesame“genome”entry. Itisalsodifficulttoeasilysearchforcompleted
genomes, ratherthandraftswithhundredsofcontigs; evenmetagenomicprojectsareincludedas
a single entry in both “Genome” and “Assembly” databases. Even more difficult is to search by
sequencingtechnology,duesometimestothelackofinformationortotheuseofdifferenttermsby
differentresearcherswhensubmittingtodatabases. Thus,whilewehavetriedtoensuretheaccuracy
ofthenumberspresentedinthefollowingparagraphsandFigures2and3theyarenotintendedtobe
anabsolutelyprecisedescriptionofthedatabasescontent.
Attheendof2015,asearchfor“actinobacteria”intheNCBIdatabases(searchstring“txid1760
[Organism:exp]”) identified 1065 genomes and 7057 assemblies. Of these, 83 genomes and 4268
assemblies (over 60%) belong to Mycobacterium species alone, mostly to clinical isolates of M.
tuberculosis(with3635assemblies). Sincethisreviewfocusesonactinobacteriarelevantfornatural
productsresearch,genomesandassembliesfromspeciesbelongingtotheclinically-importantgenera
Mycobacterium(TaxonomyID:1763),Propionibacterium(TaxonomyID:1743),Gardnerella(TaxonomyID:
2701),andCorynebacterium(TaxonomyID:1716),andspeciesofBifidobacterium(TaxonomyID:1678),a
genusthatisbecomingmoreabundantindatabasesbecauseoftheprojectsonhumanmicroflora,were
filtered-outfromthesearches;thisisnotbecauseoflackofpotentialrelevancetonaturalproducts
researchbutbecausethelargenumberofassemblieswouldintroduceamisleadingbiasintheanalysis
reportedhere. Theresultwasthatthereare849genomesand2073assembliesofactinobacteriastrains
potentiallyrelevanttonaturalproductsresearchavailableintheNCBIdatabases(Figure2). Ofthe
2073assemblies,1865assembliesareonlycompletedtocontigorscaffoldlevel.
ThePATRICdatabase[48]searchtoolallowsamorecomprehensiveandprecisesearchofbacterial
genomesand,importantly,includesthecompletionachievedandsequencingtechnologyused. Atthe
timeofwriting,thenewestentriesinthePATRICdatabaseweredatedNovember2015,andcontained
7148genomeassembliesofactinobacteria(taxonID1760). Almosthalfcorrespondedtoclinicalisolates
ofMycobacteriumtuberculosis(3785)andatotalof5330correspondedtotheclinically-relevantstrains
of Mycobacterium, Corynebacterium, and the increasingly represented Gardnerella, Propionibacterium,
Mar.Drugs2016,14, 78 8of19
andBifidobacteriumgenera. Asexplainedabove,inordertoprovideamorefaithfulrepresentation
of the genome sequencing projects relevant to natural products research, all assemblies of strains
belongingtothesegenerawerediscardedfromthestatisticsshowninthisreview;whilemanyofthese
actinobacteriamightberelevanttonaturalproductsresearch,thelownumberofactualspeciesfiltered
outisnMoatr.l iDkrueglsy 20t1o6, i1n4,fl xu encetheconclusionsofthisreview. 9 of 20
A

Technology Sanger 454 Illumina IonTorrent PacBio SOLiD
Year 2007 2 2
2008 3 3 1
2009 9 12 4
2010 4 22 2
2011 5 14 7
2012 9 24 27 3 2 0
2013 8 33 286 5 2 2
2014 11 50 546 7 12 0
Nov 2015 6 36 274 89 23 0

B

Technology Sanger 454 Illumina IonTorrent PacBio SOLiD
Year 2007
2008
2009
2010 11 13 2
2011 2 27 16 1
2012 4 84 85 1 2 1
2013 8 35 292 1 3 1
2014 8 35 529 0 10 0
Nov 2015 1 51 502 82 7 1

FigureF3i.gDuries t3r.i Dbuisttrioibnutoiofna osfs easmsebmlibelsieisn inclculuddeedd iinn tthhee PPAATTRIRCI C(A() Aan)da nNdCBNI C(BB) Id(aBta)bdasaetsa abt athsee senadt theend
of2015o,fa 2c0c1o5r, daicncogrdtoingth teo ttheec htencohlnoogloygyu suesded.. NNoottee tthhaat tsosmome aessaesmsbelmiesb lhiaevseh baeveen bobeteanineodb twaiitnhe ad witha
combincaotmiobninoaftiosenv oef rsaelveteraclh tnecohlnooglioegsieasn adndh haavvee bbeeeenn aacccocounutnedte fdorf oinr eianche aocf hthoemft. hPerimor. tPo r2i0o0r7 ttohe2r0e 07there
are 18 assemblies not included in the figure because the sequencing technology is not stated,
are18assembliesnotincludedinthefigurebecausethesequencingtechnologyisnotstated,although
although most certainly all were obtained by Sanger sequencing, or at least the 15 with complete
mostcertainlyallwereobtainedbySangersequencing,oratleastthe15withcompletestatus.
status.
PATRICallowsamorecomprehensiveperspectiveontheuseoftechnologiesthanNCBI(which
seemsnottocontainsearchableinformationpriorto2010);however,itisstillnoteasytoanalysethe
Mar.Drugs2016,14, 78 9of19
information(thenamesusedvary,e.g.,454canbereferredtowithjust“GSFLX”)andmakeanysearch
andsortingverydifficult,andtherewere118genomessequencedwithmorethanonetechnology,
almost always involving 454 and Illumina, and there were over 300 entries without an associated
technology)and,consequently,wechosetodownloadtheActinobacteriagenomestableandanalyse
itmanually. Thepicturepainted byouranalysis(Figure 3)showsthatIlluminais byfarthemost
commonlyusedtechnology,duetothelowcostpernucleotide,theveryhighthroughput,andthe
wideavailabilityofsuppliers. Asmentionedabove,thelongerreadsof454pyrosequencingmadethis
technology,despiteitshighercost,thechoicewhenamorepreciseassemblywasdesired,inparticular
forPKSandNRPSgeneclusters, andsoitwasthemaintechnologyuseduntiljusttwoyearsago,
whenIlluminatookoff. AcontinuouspresenceofSangersequencingreflectstheuseofthistechnology
tocomplementthelimitationsofNGSwhenthegoalistoobtainafinishedgenome;end-sequencing
ofgenomiclibraryclonesandsequencingofPCRampliconstofillgapsintheassemblyarethemost
commonapplicationsofSanger. SOLiDtechnologyhasbeenaveryminorplayerwithonly2–4entries
inPATRICandNCBI,respectively. Interestingly,IonTorrentseemstohavegainedmomentuminthe
pastyear;sincemostofthesubmittedprojectsoriginatedatthesameinstitution,thismaynotreflect
wideradoption,althoughimprovementsinlibraryconstructionwithhighmol%G+CDNA[49]and
bioinformatics[50]mighthelpthistechnologytobemorewidelyembracedinthefuture.
AssembliesobtainedusingTGSPacBioweresubmittedtothedatabasesfrom2012,butthefirst
examplesmadeuseprimarilyofIlluminaand454SGS,usinglimitedcoverageobtainedwithPacBio
forscaffoldingofcontigsandgapclosing[51,52].
7. PacificBiosciencesSMRTPlatform
The first streptomycete genome that appears in the literature as sequenced with PacBio is an
unfinished sequenceof Streptomyces sp. strainMg1 [53]; despite therelativelylow coverage (20ˆ)
mostofthechromosomewasobtainedasasinglecontig(accessionGCA_000412265.1);however,the
authorsdidnotpresentadetailedanalysisandonlyhighlightedtheimprovementofthesinglecontig
asopposedtoaprevious466-contigsassemblyreleasedbytheBroadInstitute(ABJF00000000)using
Illumina;amoredetailedcomparisonofbothassemblieswasincludedinareviewarticlebyHarrison
andStudholme[54]but,sinceIlluminatechnologyhadevolvedenormouslyduringthefiveyearsthat
separatebothassemblies,thecomparisonmaybemisleading.
OurgrouphasrecentlypublishedthesequencingofthegenomeofStreptomycesleeuwenhoekiiC34,
astrainisolatedfromtheAtacamaDesertinChile[43]. Thegenomewassequencedandassembled
independently with PacBio and Illumina MiSeq (paired-end 250 nt reads) between August and
November 2013. In addition, PAC clones from a genomic library were also sequenced with 454
Junior[55],andwealsohavesequencinginformationobtainedwithSangerduringthestudyofspecific
geneclusters([55]andRazmilic,manuscriptinpreparation). Thesedata,obtainedatalmostthesame
time,provideausefulcomparisonoftherelativestrengthsandlimitationsofallthreetechnologies;
someofthesewerealsodiscussedin[43]butamoredetailedviewwillbeprovidedhere.
8. PKSModularitycanbeResolvedwithPacBio
The genome of S. leeuwenhoekii C34 was first assembled by Busarakam and co-workers using
Illumina100ntpaired-endreadsinto658contigstotalling7.86Mb[56];analysisofthisdraftrevealed
numerousmiss-assemblyissuesinthePKSgenes,notsurprisingbearinginmindtheshortreadlength
used[43,55]. WealsosequencedthisgenomewithPacBioRSII.Afteraquality-filterofthereads,we
obtainedalmost1Gbofsequencedatafrom2SMRTcells(plusafirstcellthatfailedandgenerated
only77Mbofsequence,sothefullassemblywasessentiallyderivedfromjust2SMRTcells)which
wasassembledintojust3contigsof7.9Mb,95kband10kb. Oddly,thesmallestcontigmatcheda
stretchofthelargestcontigwithover90%identity;thesesmallanderror-pronecontigshavebeen
observed in other PacBio sequencing projects (e.g., [57]). The largest contig contained an almost
Mar.Drugs2016,14, 78 10of19
completechromosome,inasingleuninterruptedsequencewithoutthe“Ns”typicalofscaffolding
withIlluminasequencing;onlytheendsoftheterminalinvertedrepeatswerenotfullysequenced.
Almostconcomitantly,wesequencedthisgenomewithIlluminaMiSeq250ntpaired-endreads,
obtained1.25Gbofdataofwhich712Mbwereassembledinto279contigs,mergedinto175scaffolds,
totalling8.1Mb. ThisassemblyhadastrikingsimilaritywiththePacBioassembly,andmostofthe
studiedPKSgenesfittedtheexpectedmodularity([55]andunpublisheddata). However,evenwith
suchhighcoverage,somePKSmoduleshadbeenmisassembled[43].
9. IdentificationofCircularorLinearReplicons
The assemblers produce linear molecules, but do not make decisions on the topology of the
DNA molecule. Due to the long reads of PacBio, some reads will run into the terminal inverted
repeats(TIRs)oflinearreplicons,allowingtheidentificationofatleastthebeginningoftheTIRs[43];
similarly,thepresenceofdirectrepeatsattheendsofthecontigwillundoubtedlyindicatethatthisisa
circularrepliconandallowthecircularisationofthemoleculetoremovetheduplicatednon-existent
sequence[43]. Illuminadidanexcellentjobatassemblingthecircularplasmidintoasinglemolecule,
butwithoutthedirectterminalrepeatofthePacBiocontigitwouldnothavebeenpossibletoidentify
itasacircularmolecule. AnotherinterestingfindingwasthatPacBiocouldreadthroughstretchesof
sequencedifficultorimpossibletoreadevenbySangersequencing,apparentlyduetotheformation
ofcomplexsecondarystructures(FigureS2).
10. LimitationsofPacBio
10.1. InsertionsandDeletions: ShiftsintheReadingFrame
ThemainknownlimitationofPacBioinactinobacteriaistheresolutionofGorChomopolymers;
inourexperience,thefinalconsensussequencetendstomissaGorC,perhapsreflectinganissuewith
theassemblyalgorithmratherthanalimitationofthecurrentsequencingchemistry;ifso,itmightbe
possibletofixthisinfuturereleasesofthesoftware. Weobservedtheoppositeproblemwiththe454
technology,wherewehaveobservedaconsistentinsertionofaGorCinhomopolymers(FigureS1).
Inbothcases, theinsertionordeletioncausesashiftinthereadingframewithinaproteincoding
sequence(PCS),whichiseasilyidentifiablebystudyingthe“GCFrameplot”. Duetothehighmol%
G+CofactinobacterialDNA,thereisabiasednucleotidecompositionateachofthethreepositionsof
acodoninaPCS:GorCarepresentatthethirdpositioninover90%ofcodons,whiletheyareinthe
secondpositioninonlyaround50%ofcodons,andinthefirstpositioninabout70%ofcodons[58].
ThesoftwareFRAMEplotsthreelinescorrespondingtothemol%G+Cofeachofthethreepositionsin
thecodonscontainedwithinaselectedwindow-size[59];achangeinthedistributionofthethreelines
(acrossingoflines)withinaproteincodingsequenceisindicativeofashiftinthereadingframecaused
byaninsertionordeletion(Figure4). Frame-shiftsareusuallyeasytoidentify,unlesstheyoccurat
thebeginningorendofthePCSwithastartorstopcodoninproximity. Iftheencodedproteinshows
closely-relatedhomologousproteinsinthedatabases,aBLASTsearcheasilyidentifiesframe-shiftsas
wellandcanbeusedtoconfirmtheFRAMEplotanalysis.
TheinsertionsanddeletionscanbecorrectedbymeansofdirectedPCRamplificationandSanger
sequencing(Figure4)oratagenome-widescalebydeep-sequencingwithIlluminaandmappingof
readstothePacBioassembly[43,60,61]. Inanycase,theyareeasilyaddressableanddonotposesuch
aproblemasmisassemblyofPKSmodules.
10.2. SequenceMissingfromtheFinalAssembly
ThemoststrikingfindingduringcomparisonofourPacBioandIlluminaassemblieswassequence
missingfromthePacBioassemblythatwaspresentinthepre-assembleddata. Duringsequencingof
theS.leeuwenhoekiiC34genomewefoundthatover130kbofsequence,mostlikelyrepresentinga
linearplasmid,waspresentintheIlluminabutnotthePacBioassembly;however,thissequencewas