Original citation:
Jarvis, Stephen A., 1970-, Mirsky, J. S., Peden, J. F. and Saunders, N. J. (2000)
Identification of horizontally acquired DNA using genome signature analysis. University
of Warwick. Department of Computer Science. (Department of Computer Science
Research Report). (Unpublished) CS-RR-379
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using genome signature analysis
Stephen A. Jarvis
1
, Jason S. Mirsky
1
, John F. Peden
2
and NigelJ. Saunders
3y
ProgrammingResearh Group, OxfordUniversity ComputingLaboratory
1 ,
University of Oxford,Oxford OX1 3QD,UK.
MRC MoleularHaematology Group
2
,Moleular Infetious Diseases Group
3 ,
Institute of MoleularMediine,
University of Oxford,Oxford OX3 9DS, UK.
September1, 2000
Abstrat
Motivation:Identiationofregionswithuntypial
perentageG+Compositionanddinuleotide
signa-turesaretwogenomeanalysistehniquesusedinthe
identiation of horizontallyaquired DNA.We
de-sribe a generi program framework for performing
bothtypesof analysisin linear time. The approah
isextendedforlength>2oligonuleotidesignatures.
Usingthe derivedprogram wetest someofthe
on-lusionsofKarlinandBurge,theprimaryexponents
ofthesetehniques.
Results: Wedemonstratethat nosinglemethod of
signatureanalysisissuÆientfortheomplete
identi-ationofhorizontallyaquiredDNA.Consequently
weprodueafastprogram-andarobust
methodol-ogy-fortheprodutionandemploymentgenome
sig-natures intheidentiation ofhorizontallyaquired
DNA.
Availability: Softwareavailable fromrstauthor.
Keywords: DNA;genome;signature;algorithms.
now at: Department of Computer Siene,
Uni-versity of Warwik, Coventry CV4 7AL, UK. Email:
stephen.jarvisds.warwik.a.uk. To whomorrespondene
shouldbeaddressed y
urrentaddress: SirWilliamDunn Shool of Pathology, UniversityofOxford,Oxford. OX13RE
Introdution
In addition to the vertial transmission of adaptive
mutations, horizontal transferof genes between
un-relatedspeiesisinreasinglyreognisedasan
impor-tantomponentof evolution[Doolittle,2000℄. Most
ifnotallspeiesinludematerialwhihhasbeen
a-quiredinthisway. Thesopeforthistypeofgeneti
exhangeislimitedin higherorganismsinwhihthe
germellsare sequesteredand are notnormally
ex-posedtoforeignDNA.However,inbateria,inwhih
theellsthatformtheoriginsofnewpopulationsare
exposed to DNAin the environment,horizontal
ex-hangeofDNAisanongoingandimportantproess
[Arber, 1993, Lawrene et al., 1998, Maddox, 1998,
Woese, 1998, Brown and Doolittle, 1999, Doolittle,
1999, Forterre and Philippe,1999, Jainet al., 1999,
Martin,1999,Nelsonetal.,1999,Faguyand
Doolit-tle, 2000, Moreire,2000℄. For example, it has been
estimatedthat18%ofthegenesin E.olihavebeen
aquired by horizontalexhange sineits divergene
fromSalmonellaspp.approximately100millionyears
ago[Lawreneetal.,1998℄.
Horizontal exhange is reognised as being
im-portant in the evolution of baterial virulene, the
most widely aknowledged examples of whih are
har-omposition whih diers from that of the rest of
thegenome; terminal inverted repeats; proximity to
tRNAgenes [Hou,1999℄and assoiationwith
trans-posases whih may be intat or present as
rem-nants. These islands often ontain groupsof genes
withfuntionswhihanreadilybeinvokedin
mak-ing a speies more virulent, suh as toxin
pro-dutionandseretion[GroismanandOhman,1996,
Hakeretal.,1997, Alfanoetal.,2000℄. Usuallythe
origin of the transferred DNA is not known and
the soure of these genes - so signiant in
vir-ulene - remains an interesting enigma. In a
small number of instanes, however, the likely
ori-gin of horizontally transferred genes an be
in-ferred with some ertainty [Vasquezetal.,1995,
Krolletal., 1998,Saundersetal.,1999℄.
In order to beome established within abaterial
population, an aquired gene (or other gene with
whihitisassoiated)mustleadtoaninreasein
ba-terialtness. Thistakesplaethroughoneormoreof
thefollowingfators: inreasing transmissibility,
in-reasingthesizeoftheolonisingpopulation,
estab-lishing new nihesin whih theorganism an grow,
orevadinghostimmune responses. Thisinreasein
tness must then be suÆient for the sequene to
beome prevalent in the population to whih it has
beentransferred. This ours either through lonal
replaementofthepreviouspopulationthatdidnot
havethegeneorbyfurtherhorizontaltransferofthe
gene within the new speies to whih it has been
transferred. Theproesseswhih mightbeexpeted
tobeinvolvedininreasedtnessarebroadlysimilar
to those involved in virulene. It is therefore
im-portant to be ableto identify sequenes whih have
beenaquired byhorizontal transferin order to
un-derstandtheevolutionaryhistoryofaspeiesand,in
theontext ofbaterialpathogens, toidentify genes
potentiallyassoiatedwithvirulene.
DNAfromdierentgenetibakgrounds(e.g.,
ba-terialspeies)hasdierentharateristis. Theseare
aprodutoftheodonswhiharepreferentiallyused
bythepartiularspeiesandtheenvironmental
on-ditions in whih itgrows,aswellasthetypesof
er-rortowhih theDNArepliationenzymesareprone
and dierenesin theabilityofeahenzymeto
or-tions of DNA whih are derived from dierent
ge-neti bakgroundsby means of theiruntypialbase
omposition. DNA whih is inorporated following
horizontaltransferwillbeexposedtothesame
pres-sureswhihgavethereipientitsharateristi
om-position. As a result, over time the aquired DNA
will begin to onform to the average DNA
ompo-sition of the reipient by a proess alled
amelio-ration [LawreneandOhman,1997℄. However, the
rateofameliorationissuÆientlyslowthatitis
pos-sibletoidentifyregionsofDNAwhihhavebeen
hor-izontallyaquired. Theextenttowhihthissequene
isuntypialthereforedepends onboththeextentof
thedierenebetweenthedonorandreipientspeies
andtheperiodoftimewhihhaselapsedsine
aqui-sition.
Therstmethodfortheidentiationofregionsof
potentiallyhorizontallytransferredDNAisbasedon
%G+Charateristisof the sequene. This is
use-ful beause the %G+C within prokaryoti genomes
is relatively homogeneous within a speies but
dif-fers between unrelated speies [Karlinet al.,1998℄.
Signiantly dierent %G+C ontent in setions of
DNAtherefore indiates possible horizontal
aquisi-tion[Karlinetal., 1998℄.
Experimental studies showed that the
dinu-leotide odds ratios were similar in most
or-ganisms for the bulk of their DNA and
essen-tially the same for dierent parts of the same
genome[Josseetal.,1961℄. Lookingatsequenes
de-rived from dierent sequene bakgrounds revealed
thatmoreloselyrelatedspeiestendedtohavemore
similar dinuleotide ompositions than unrelated
speies [Karlinet al.,1994.a, Karlinetal.,1994.b,
Karlinet al.,1994.℄.
Karlin and his olleagues noted that the speies
dierenes in base usage were more omplex and
that, in addition to dierenes in %G+C
omposi-tion,dierentspeies usedharateristipatterns of
dinuleotide pairswithin theirgenomes. This
obser-vation provided the basis for the seond approah
in whih the DNA is seen not as a string of
sin-glebases butasasequeneofpairsofdinuleotides:
the sequene ACGTG, for example, would be
GTand TG.The perentage of eah of the 16
din-uleotide pairs was found to dier between speies
andalthoughthereasonsforthesedierenesarenot
fully understood, this provides aseond method for
identifying horizontallyaquired DNA.The method
is known as dinuleotide signature (DNS)
analy-sis[KarlinandBurge,1995℄.
Inthispaper:
Karlin's approah to %G+C ontent and DNS
analysisisexaminedandextensionstohis
meth-odsareproposed.
Ageneriprogramforthelinearomputationof
n-oligonuleotidesignaturesisdeveloped.
Karlin and Burge's observation that
n-oligonuleotide signatures are highly implied
by the DNS is onrmed. However, it is also
demonstratedthat ifdierentwindowsizes and
word lengths are used, n-oligonuleotide
signa-tures point to dierent, potentially relevant,
areasfrom theDNSgraphs.
A omparison is madeof the dierent methods
foridentifying horizontallyaquired DNA;
on-lusionsaredrawnandsupportingresults,whih
desribehowthemethods anbeused,are
pre-sented.
Systems and methods
Computational analysis
Asymptotianalysis is away ofomparing the
per-formaneofdierentomputationalalgorithms. This
omparison is used to selet oneomputational
ap-proah,whih mayprovideamoreeÆientsolution,
overanother.
Thebig-O notationdesribes theupperbound of
theasymptotigrowthofafuntion. Forinstane,if
analgorithmtakes
1 3 n
2
2n+4steps,itisofO(n 2
),
sineit is bounded from above bya funtion whih
takesn
2
stepstimessomeonstant[Kaldewaij,1990℄.
Karlin'smethodsforndinghorizontallyaquired
DNArelyonomparingDNAwithitselfbymeansof
(bounded byO) time
O(log2n) 0:1seonds
O( p
n) 5seonds
O(n) 1hour15minutes
O(n 2
) 450years
Figure1: Lengthoftime nomputationstakewhen
n=5;000;000andasingleomputationtakes1
mil-liseond. Tableadapted from[Kaldewaij,1990℄.
mathematialalulation. Thehromosomesof
ba-teriaare typiallyomposed of0.5 to5millionbase
pairs (Mb). Performing alulations on the DNA
sequene (of length n) requires a large amount of
omputation: rst the omplete sequene statistis
are obtained, O(n); then individual segments
(win-dows) are statistially analysed and ompared with
theompletesequene,O(n
2
). Astraightforward
re-nementwouldthereforeprodueanimplementation
whih wasbounded byO(n
2 )steps.
Figure 1 shows the relationship between
asymp-totianalysisandomputationtime.
Optimisationstoprogramsanofoursebemade
and omputers work at a far greater rate than the
examplesuggests. However, thetable demonstrates
thatadiereneintheorderofomplexityofan
algo-rithmisdiretlyproportionaltotheruntime.
Redu-ing an algorithm from an O(n
2
) solution to a O(n)
solution will make a onsiderable dierene to the
number(andvariety)ofresultswhihanfeasiblybe
obtained.
Methods of genome analysis
%G+Content
Calulating the %G+C ontent of an organism's
DNA is a simple method for nding regions of
ge-neti interest. %G+C ontent soresare alulated
for segments (windows) of DNA by adding the
fre-queny(numberdivided bywindowlength) ofG to
thefrequeny of C. A plot of %G+Content is
de-rivedbyslidingthewindowovertheompletegenome
Dinuleotide signatures an be used to show areas
where pairs of bases are lustered more frequently
thaniftheywere distributed arossthesequeneby
hane. A DNS graph is based on a alulation of
odds ratios for eah of the 16 dinuleotides. This
odds ratio (probability) is alulated by taking the
frequenyofadinuleotideanddividingitbythe
ex-petedprobabilityofndingthedinuleotide within
apartiularsequene:
p xy = f xy f x f y (1) f x
is thefrequeny of nuleotide x within the
se-quene and f
xy
is the frequeny of thedinuleotide
xywithin the(irular)sequeneofDNA:
f xy = #xy #dinuleotides (2)
where#dinuleotides=SequeneLength 1
p xy
represents the odds ratio for dinuleotides
in single-stranded DNA. To alulate the odds
ra-tio for double-stranded DNA, written
p
xy
, the
se-quene and its reverse omplement are
onate-nated [Burgeetal.,1992℄. The alulation remains
thesame,butwithasequenetwiethelengthofthe
original.
To nd areas of possible horizontally aquired
DNA,awindow(f)(ofsomehosensize{50Kbfor
example [KarlinandBurge,1995℄) is reated. The
dinuleotide probabilities of this window are then
ompared with the dinuleotide probabilities of the
overallsequene(g). This isdonefor eah ofthe16
dinuleotides and the normalised results are added.
The result is an overall dinuleotide dierene
be-tweenthewindowf andthesequeneg.
Æ
(f;g)=( 1 16 ) 16 X xy j p xy (f) p xy
(g)j (3)
Usingthesamemehanisasthoseusedin%G+C
ontentsignatures,KarlinandBurgesuggest
repeat-edly sliding the window one position along the
se-queneandrepeatingthedierenealulationuntil
dows. Graphing the results shows the regions of
largestdierene,whihareandidatesforhorizontal
aquisition.
Oligonuleotidesignatures
Justasgreaterinformationandresolutionisobtained
when dinuleotide sequene omposition is
onsid-ered rather than single base omposition,
analyz-ing sequenes on the basis of even longer motifs
may provide additional useful information.
Trinu-leotide omposition addresses odon usage in
ex-pressedportionsofopenreadingframes. However,it
maybethat preferenesforpartiularpairsormore
of odons, mutational proesseswhih favour or
se-let againstlongersequenes, orproesseswhih
af-fet the omposition of the non-transribed strand
willinuenethesequeneompositioninadditional
ways. The method has therefore been extended so
that signatures an be determined for longer
mo-tifs[Karlinetal.,1997,Mirsky,1999℄.
Dinuleotide analysis is easily extendible to
oligonuleotidesignaturesoflengthn.
Æ
(f;g)=( 1 4 l ) 4 l X i:s j p i (f) p i
(g)j (4)
where lis thelengthof oligonuleotideswhih
om-prisethesignature,sisthesetofallpermutationsof
lengthlandi isonesuhpermutation.
Inaddition,thevarianev andstandarddeviation
sdanbedetermined forthe absolutedierenes of
thepvaluesateahpointandfortheÆvaluesaross
theentire sequene.
v= n P x 2 ( P x) 2 n 2 (5)
To alulate thevariane for
Æ, eah
Æ
value
or-respondsto ax andthe numberof
Æ
values(the
se-quene length) orresponds to n. To alulate the
variane of the p values, x = p and n = 4
l
. The
standarddeviationissimplythesumofthevariane
values,sd=
Whenthe entire populationis notknown, that is
ifthewindowslidesbymorethanonebasebetween
alulations,anestimateofthevarianeisalulated:
v=
n
P x
2 (
P x)
2
n(n 1)
(6)
Implementation
The same proedure is used to implement %G+C
ontent, dinuleotide and n-oligonuleotide
signa-tures. Thesliding windowimplementation (or
simi-lar)is mehaniallyequivalentforeah;theprimary
dierene between eah signature is simply the
for-mulausedtoalulate thepoints.
Ageneriimplementationis explored.
%G+Contentrequires the smallestspeiation
and is therefore most appropriate to illustrate the
derivation oftheprogram. An O(n
2
) solutionis
de-rivedand,byamethodknownasloopattening,this
isonvertedintoanO(n)solution.
TheO(n)implementationprovidesthebasisfor a
omparativestudy ofthese methodsfor the
identi-ationofhorizontallytransferredDNA.
Generi genome signatures
The speiation of a program for %G+C ontent
analysis (GC
prog
) is presented in the guarded
om-mandlanguage[Kaldewaij,1990℄.
P =fN1g
[ on N: intfN1g; A:array[0..N) of DNA;
on WS:int f1WSNg;
var n: int;
GC prog ℄.
Q=fr=8p:0p<N :8q:0q<WS:
g=(#i:pi(p+q):
A:(imodN)=C
_A:(imod N)=G)=WS)g
Thespeiationontainsthreeparts:the
preon-dition P; the part in the frame, denoted by square
brakets;and the post onditionQ, found after the
frame.
Window 1 2 3
Movement of sliding window
Nucleotide leaving window
Nucleotide entering window
Old window
New window
GC(window’) = GC(window) - 1
If ( n = C or n = G )
Sequence
of circularity of bacterial DNA
before termination because
Windows wrap round
If ( n = C or n = G )
GC(window’) = GC(window) + 1
A.
B.
C.
Sequence
ws
0 <= ws < WS
Window
C
A
A
T
G
Figure 2: Derivationof ageneriDNAsignature
al-gorithm: A. Generate a window for every position
in the sequene by sliding the window through the
sequene,theouter loop; B.Calulatesignaturesfor
eah window, the inner loop; C. Flatten the inner
loopforO(N)solution.
Thepreonditionstatesthatthesequenemustbe
ofatleastlength1(for%G+Contentthisis
appro-priate;forlargeroligonuleotidesthisminimummust
bethelengthoftheoligonuleotide).
Theframeontainstheinformationrelevanttothe
developmentof the program: the onstant N is the
length of the DNA sequene A; WS is the window
size;nis avariableused fortraversingthesequene
andGC
prog
issimplyamarkerfortheprogramitself.
Thepostondition desribesthe resultr, alist of
%G+Contentdierenes, onefor eahof the
win-dowsinthesequene. Themodulo(mod)isontained
withinthespeiationtoaountfortheirularity
ofthebaterialDNA.Itisalsonotedthattheresults
list r is written to a le asthe program proeeds
-thissavesonRAM.
Toalulateawindowfromeverypositionin the
se-quene,thewindowslides throughthesequene (f.
Karlin). This outer loopis determined byreplaing
theonstantN bythevariablenandusingthe
post-onditionQfn =Ng. This suggestsstarting nat 0
and setting theloop guardto n6=N. This oers a
solutiontotheouterloopofO(N). SeeFigure2-A.
Derivation: stage 2
The inner loop is now alulated. The inner loop
orrespondstothealulationforonewindow. Sine
the inner loopwill be plaedwithin the outer loop,
panbereplaedbynintheinnerloopspeiation.
[ varws,g: int;
ws,g=0,0;
W prog ℄.
Q i
=ft=8q:0q<ws: g=((#i:ni<(n+q):
A:(imodN)=C
_A:(imod N)=G)=WS)g
TheinnerloopisderivedbyreplaingonstantWS
withvariablewsandsplittingtheinvariantintotwo
hunks,onefor alulatingGontentand theother
for alulating C ontent. The inner blok is also
O(N). SeeFigure2-B.
Sine the windowsrange from size 1 to N 1, a
mathematialaverageforthewindowsize isN=2.
ThisgivesO(WS)[WSn(N=2)℄,i.e.,O(N)forthe
in-ner loop. As this ours O(N) times, one foreah
iterationoftheouterloop,theomplexityofthe
al-gorithmisO(N
2 ).
Derivation: stage 3
Itispossibletoattentheinnerloopfromanaverage
size of N=2 to a onstant of 2. Instead of simply
ounting and disarding the number of Gs and Cs
in eahwindow,itispossibletousetheinformation
fromthepreviouswindowtoalulatethenext. This
is possible bysubtrating theitems whih leavethe
eah timethewindowslides. SeeFigure2-C.
To alulate the new inner loop we now split the
loop into twoparts: the rst alulates the ontent
for the rst window, O(N) one only; the seond
alulates the remaining N 1 windows, onstant
2O(N) = O(N). As we are adding terms rather
thanmultiplyingthem, thenewalgorithmisO(N).
Computing other signature types
The derived algorithm is generi in the sense
that it provides the neessary O(N) framework for
whihever type of signature one is trying to
pro-gram. The inner loop alulation (for %G+C
on-tent) an simply be replaed by the formula for
n-oligonuleotidesignatures[Mirsky,1999℄.
Oneinterestingobservationshouldbenotedfor
n-oligonuleotidesignatures.
Whennuleotidesenterandleaveawindow,some
adjustment needs to be made to the tallies for the
oligonuleotidesinthenewwindow. InFigure2part
C, for example, a ount of the dinuleotides in the
window requires 1 to be subtrated from the CA
ountand1to beaddedtotheTGount.
Thisis aneasyalulationto make,yet onemust
be areful about storing and aessing a list of
oligonuleotideourrenes. Ifalinearsearhisused
then the omplexity of the overall algorithm may
hange;if4
l
>N,thegenerisignatureprogramwill
notmaintainO(N).
Tooveromethis problemahashfuntion is used
to alulate the array position of any permutation.
DNA is oded as an enumeratedtype, where A=0,
T=1,C=2,G=3. Thehashfuntion isalulatedas
follows: if the oligonuleotide beingsearhed for in
the hash table is ATCA, the hash table osetis as
follows:
04
3
+14
2
+24
1
+04
0
=0+16+8+0=24
The ourrene ount for this oligonuleotide is
thereforefoundatposition 24inthehashtable.
Thealulationisalwaysunique,so searhingthe
hashtable is neverneessary. Itis alsoonstant,so
Using large windows the HNS (part b, bottom) is
highlyimpliedfrom theDNS(part a,top).
Thismethodalsohastheadvantageofnot
requir-ing any of the oligonuleotide permutations to be
stored textually, and thus wasting memory. Eah
oligonuleotide permutation is simply a formula
basedonthenuleotideontentandtheenumerated
typevalues.
One last optimisation an be made. This
imple-mentationisforÆandnot
Æ
,asthealulationisfor
single-stranded DNA. It is possibleto write a
fun-tion whih derives the omplement of the sequene
and onatenates it onto the end, as suggested by
KarlinandBurge(1995)andoriginallyimplemented
by Burge et al. (1992). This turns N into 2N.
WeprovethatÆatuallyequals
Æ
,eventhoughp6=
p .
TheproofofthisequivaleneisillustratedbyMirsky
(1999). The50%gainreduestheomputationtime
ofaderivedprogramstillfurther.
size 1 Kb. The rst graph shows the DNS; the
seondshowstheHNS.
Disussion
Comparative results
KarlinandBurge(1995)presentaDNS graphofH.
pylori; this graphis reproduedby ourderived
pro-gram andis shown inFigure 3-a. Karlin andBurge
doument that signatures reated from longer
per-mutations are \highly implied" by the DNS graph.
Thelength6-oligonuleotidesignature graph(HNS)
for window size 50,000 (Figure 3-b) is indeed
im-plied by the DNS results, although the results are
not the same. The observation of impliation does
notholduniformlyforother windowsizes. Dierent
areasofinterestarehighlightedasthewindow-length
and permutation-lengthvariables aremodied
(Fig-ure4).
The results also show that
Æ
averag-valuabledata. Bystudying P xy
p xy
(dinuleotide
probabilityforthewholesequene minusthe
proba-bility foran individualwindow)for allxy
permuta-tionsoflength2,regionsofimportaneappearwhih
areaveragedoutfrom the
Æ
graphs.
The graphsfor dierent permutation lengths an
be ompared. This is beause signatures are based
on odds ratios and so the number of permutations
is absorbed into the ratio yielding absolute results.
Signiant results are identied by seletion based
onstandarddeviationthresholdsfromthemean. The
mostappropriatethresholdsshouldbedeterminedon
anorganism-by-organismbasisandonthenature of
thestudybeingperformed,andremaindependenton
windowsize andpermutationlength.
DNS analysis using a 50,000 base window has
previously been used to identify regions that have
been horizontally aquired in N. meningitidis,
us-ing a threshold for detetion of 3 standard
devi-ations [Tettelin etal.,2000℄. Analysis of the H.
pylori sequene, using the same parameters the
DNS analysis,identied tworegionsinluding
read-ing frames HP0412 to HP0465 and HP0497 to
HP0573. The seond region ontains the ag
pathogeniity island previously identied using this
method[KarlinandBurge,1995℄.
Usingthesamethresholdof3standarddeviations
theHNSidentiedonlyasingleregion(fromHP0499
toHP0578)ontainingtheseondregionidentiedby
theDNS. Usingareduedthresholdof greaterthan
2 standarddeviations the HNS also identied a
re-gioninludingthe rstidentied by theDNS (from
HP0409toHP0480),andanadditionalregion
inlud-ingreadingframesHP0974to HP1030.
OnediÆultywiththis approah isthatthelarge
sizeofthewindowsmeansthatthismethodonlygives
a general indiation of where the atypial sequene
is loated. In the ase of the ag pathogeniity
is-landtheregionwhihhadbeenhorizontallyaquired
was dened by a judgement based upon
interpreta-tion of the oding regions [KarlinandBurge,1995℄.
In the ase of N. meningitidis, in whih a similar
analysis was performed, the limits of the regions
thathadbeenhorizontallyaquiredweredetermined
strain[Tettelin etal.,2000℄.
Usingawindowlengthof1000,DNSanalysis
iden-tied177windowsof>3standarddeviationsabove
themean. Theseorrespondto25regionswith
on-tiguousORFsand2regionswhihdidnotontain
an-notatedodingregions. TheHNSanalysis identied
a broadly similar number of regions, with 222
win-dows>3standarddeviationsabovethemean. These
orrespondto32regionswithontiguousORFs(and
none notontaining any annotatedoding regions).
However,althoughtheanalysesbasedupondierent
length motifs identied 20 ommon reading frames
(40% of DNS and 25.3% of HNS nds) the
major-ity of the reading frames identied were unique to
oneorotheranalysis. Theuniquegenesidentied in
bothanalysesinludedthose whihare knowntobe
thesubjettohorizontal transfersuh asrestrition
modiationsystemgenes (2 identiedbyDNS and
7byHNS).Itshould alsobenotedthatgeneswhih
haveatypialsequeneompositionforreasonsother
thanhorizontalaquisitionarealsoidentiedbythis
method, inluding in this analysis the `poly E rih
protein'(HP0322)andthe`histidineand
glutamine-rihprotein'(HP1432).
Reduing thewindow size inreases the
granular-ity of the results. This is assoiated with an
in-rease in the speiity of this analysis, although
somegenesadjaenttothosethatgeneratedivergent
results will still be inludeddue to the sliding
win-dow nature of this method. Theresults onerning
theagpathogeniityislandprovedinteresting. Both
1000bpwindowsizeanalysesidentied HP0527and
HP0528(odingforagpathogeniityislandproteins
7and 8). However,theDNSalsoidentiedHP0522,
HP0523,HP0531,HP0532,HP0533andHP0534
(en-oding agpathogeniityislandproteins3,4,11,12,
13 and a hypothetial protein, respetively); while
the HNS identied HP0527 and HP0528 (enoding
agpathogeniity islandproteins15 and16) aswell
astwootherreading frameswithin theregion of
di-vergenebutnotpartofthereognizedpathogeniity
island. Theseresultsindiatethatalthoughthe
over-allpatternsofthelongerwindowanalysesaresimilar
this an be the onsequene of the presene of
analysesdier.
Karlin and Burge state that the area of
most signiane in the 50 Kb window
dinu-leotide signature points to a pathogeniity
is-land[KarlinandBurge,1995℄. Thisis supportedby
the50Kb-windowDNSand%G+Contentresults.
However, the analysis using shorter windows
indi-atesthat someofthegeneswithinthisregion,even
withintheagpathogeniityislanditself,arenot
di-vergent by1standard deviation (seeFigure 4). On
thebasis ofthesignature analysis itannot be
on-luded that all of the genes within this region have
featuresthatsuggestthattheyhavebeenhorizontally
aquired. While a1 Kbwindow implies theresults
ofthe50Kbwindow,sinethelargerwindowmerely
averages theresults of the smaller windowsit
om-prises, the reverse is not true. Examination of the
regionidentiedbythe50KbDNSusingthesmaller
windowanalysisallowsustoonludethat thelarge
peaksurrounding thepathogeniityislandis in part
identifying regions of atypial assoiated DNA, and
that the abnormal regions do not omprise all the
reognisedgenesthatomposetheagpathogeniity
island. Signatureanalysis with alargewindow(e.g.
50Kb)annotberelieduponforndingshortregions
ofatypialsequene(e.g. 2Kb)in agenome.
Reently, Lawrene and Ohman mined the
DNA of E. oli for horizontally aquired
re-gions [Lawreneetal.,1998℄. Using a odon bias
tehnique, they determined that 18% of the genes
in E.oli(approximately10%of thesequene)have
beenhorizontallyaquired sineits divergene from
Salmonellae. A similar proportion of untypial
se-queneinE.oliisidentiedusingsignatureanalysis.
A length-2 oligonuleotide signature and a length-6
oligonuleotidesignature showedthat 14%and 12%
respetively of the windows in eah were at least 1
standarddeviation abovethemean.
Conlusions
Thispaperdesribesnewsoftwareforthe
identia-tion of horizontally aquired DNA. Programs have
been developed whih perform dinuleotide and
n-oligonuleotide signatures and whih inlude
meth-standarddeviations andmeansforusein ondene
estimates. ThedevelopmentofageneriO(n)
algo-rithmfor these tasks is doumented. This has
on-siderablebenet overan O(n
2
)solution. The speed
at whih theresultingprogram exeutesmeans that
methodsofsignatureanalysisanbeomparedusing
largedatasetsandpreviousonlusionsanbetested.
Testing these signature analysis methods on
genomesequenedatahasrevealedthat%G+C
on-tent alone is not a reliable method for identifying
horizontally aquired DNA. The variane that
im-plies horizontal transfer is not always reeted in
%G+Contentandthereforewereommendtheuse
of %G+Content onlyto support data from one of
theotherdoumentedmethods.
DNS analysis does identify sequenes onsistent
with those thought to be horizontally transferred.
The generationof length-n>2 oligonuleotide
signa-tures also provides useful results. Karlin's results
suggesting that the n-oligonuleotide signatures are
highlyimpliedbythedinuleotidesignatureare
on-rmedfora50Kbwindow. However,withdierent
windowsizes, the n-oligonuleotide signatures often
point to other, potentially relevant, areas from the
dinuleotide signature graphs. In addition, we nd
that plotting individual dierenes in probabilities,
before averaging,resultsin anergranularityof
re-sults. Often an averageannot distinguish between
ahighvariationin an individualpermutation whih
isosetbylowvariationsin theotherpermutations,
ontheonehand,andmoderatedierenein all
per-mutationsontheother.
Our omparison of methods leads us to onlude
that nosingle method of signature analysis is
suÆ-ient for the omplete identiation of horizontally
aquired DNA. There is some overlap between the
results,but dierent approahesontributevaluable
additionalresultsthatanyonemethodwouldmiss.
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