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Whole genome sequencing o he black g ouse (Te ao e ix): e e ence guided
assembly sugges s as e -Z and MHC e olu ion
Wang, Biao; Ekblom, Robe ; Bunikis, Ignas; Sii a i, Heli; Höglund, Jacob
Wang, B., Ekblom, R., Bunikis, I., Sii a i, H., & Höglund, J. (2014). Whole genome
sequencing o he black g ouse (Te ao e ix): e e ence guided assembly sugges s
as e -Z and MHC e olu ion. BMC Genomics, 15(180). h ps://doi.o g/10.1186/1471-
2164-15-180
2014
RESEARCH ARTICLE Open Access
Whole genome sequencing o he black g ouse
(Te ao e ix): e e ence guided assembly
sugges s as e -Z and MHC e olu ion
Biao Wang
1*
, Robe Ekblom
1
, Ignas Bunikis
2
, Heli Sii a i
3
and Jacob Höglund
1
Abs ac
Backg ound: The di e en egions o a genome do no e ol e a he same a e. Fo example, compa a i e
genomic s udies ha e sugges ed ha he sex ch omosomes and he egions ha bou ing he immune de ence
genes in he Majo His ocompa abili y Complex (MHC) may e ol e as e han o he genomic egions. The ad en
o he nex gene a ion sequencing echnologies has made i possible o s udy which genomic egions a e
e olu iona y liable o change and which a e s a ic, as well as enabling an inc easing numbe o genome s udies o
non-model species. Howe e , de no o sequencing o he whole genome o an o ganism emains non- i ial. In his
s udy, we p esen he d a genome o he black g ouse, which was de eloped using a e e ence-guided assembly
s a egy.
Resul s: We gene a ed 133 Gbp o sequence da a om one black g ouse indi idual by he SOLiD pla o m and
used a combina ion o de no o assembly and chicken e e ence genome mapping o assemble he eads in o 4572
sca olds wi h a o al leng h o 1022 Mb. The d a genome well co e s he main chicken ch omosomes 1 ~ 28 and
Z which ha e a o al leng h o 1001 Mb. The d a genome is agmen ed, bu has a good co e age o he
homologous chicken genes. Especially, 33.0% o he coding egions o he homologous genes ha e mo e han 90%
p opo ion o hei sequences co e ed. In addi ion, we iden i ied ~1 M SNPs om he genome and iden i ied 106
genomic egions which had a high nucleo ide di e gence be ween black g ouse and chicken o be ween black
g ouse and u key.
Conclusions: Ou esul s suppo he hypo hesis ha he ch omosome X (Z) e ol es as e han he au osomes and
ou da a a e consis en wi h he MHC egions being mo e liable o change han he genome a e age. Ou s udy
demons a es how a mode a e sequencing e o can be combined wi h exis ing genome e e ences o gene a e a
d a genome o a non-model species.
Backg ound
Nex gene a ion sequencing (NGS) has spu ed a e olu ion
in he de elopmen o genomic ools o non-model
o ganisms [1]. In pa icula , sequencing comple e ansc ip-
omes [2] o complexi y- educed ac ions o genomes [3]
has enabled he iden i ica ion o genome-wide molecula
ma ke s such as single nucleo ide polymo phisms (SNPs)
and mic osa elli es (SSRs). Such in es iga ions ha e also
add essed undamen al ques ions in molecula ecology and
e olu ion, such as he genomic basis o specia ion [4,5],
mo phological a ia ion [6,7], disease esis ance [8] and
selec ion on li e his o y ai s [9,10].
A comple e genome sequence is he ul ima e genomic
ool o a species. I such a sequence is a ailable i is
possible o conduc la ge-scale, in-dep h s udies o many
impo an molecula biology p ocesses such as gene exp es-
sion, ansc ip ion egula ion, al e na i e splicing, epigen-
e ic modi ica ions and gene-p o ein in e ac ions [11-14]
which a e impo an in ecological s udies. Howe e , apply-
ing NGS echnologies such as de-no o sequencing on a
la ge euka yo ic genome is s ill a e, as i ep esen s a con-
side able in es men . The shee olume o da a gene a ed
and he compu a ional acili ies needed o assemble and
analyse i may limi he numbe o non-specialized labs ha
* Co espondence: [email p o ec ed]
1
Depa men o Ecology and Gene ics, E olu iona y Biology Cen e, Uppsala
Uni e si y, No by ägen 18D, SE-75236 Uppsala, Sweden
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2014 Wang e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed.
Wang e al. BMC Genomics 2014, 15:180
h p://www.biomedcen al.com/1471-2164/15/180
a e cu en ly able o emba k on such a p ojec . Howe e ,
mo e whole genome s udies a e needed o add ess un-
damen al ques ions on he e olu ion o genome o gan-
isa ion, such as which egions a e conse ed and which
egions change when axa di e ge and become sepa a e
species. Published whole NGS genomes o non-model
o ganisms include gian panda [15], cod [16], naked mole
a [17], macaque [18], Tasmanian de il [19], budge iga
[20], Pue o Rican pa o [21], Heliconius bu e ly [22],
Aye-aye [23], colla ed lyca che [24], as well as he 29
mammalian genomes ecen ly sequenced a he B oad
Ins i u e [25].
The la ge numbe o publically a ailable whole genome
sequences om bo h model and non-model o ganisms can
be used o aid genomic in es iga ions in ela ed o ganisms.
One app oach is o di ec ly ans e he genomic esou ces
om a model o ganism o he s udy species, which would
hen be called ‘genome enabled axa’[26]. This s a egy has
been used success ully o de elop esou ces such as mic o-
sa elli e ma ke s [27], SNPs [28], mic oa ays [29] and exon
cap u e a ays [30]. Al e na i ely, he genome sequence
om a ela ed model o ganism can be used in he assembly
o sho ead da a om he ocal species, a p ocess known
as e e ence guided (o e e ence assis ed) assembly [31,32].
He e, we desc ibe a e e ence guided, whole genome
assembly o he black g ouse (Te ao e ix, Figu e 1). We
ake ad an age o he close ela ionship be ween his
species and he well cha ac e ized chicken (Gallus gallus;
bo h belonging o he o de Galli o mes wi h a di e gence
ime o 30 ~ 40 my ) [33-35] and de elop a e e ence
guided assembly pipeline o cons uc a d a genomic
sequence. The black g ouse is well s udied as a model
o ecology and conse a ion biology [36-38] bu , un il
ecen ly, genomic esou ces o his species ha e been
la gely lacking. This wo k comple es ou genomic ools
de elopmen , p e iously ini ia ed by cha ac e izing he
ansc ip ome [39] and sequencing he majo his ocom-
pa ibili y complex (MHC) egion [40] in his species.
In heo y he e a e wo di e en ways o using he e e -
ence sequence o guide he assembly p ocess. Unde an
“align- hen-assemble”s a egy, he eads a e i s mapped
o he e e ence sequence and clus e s o eads mapping
o he same loca ion a e hen ex ac ed and assembled
de-no o. Al e na i ely, in he “assemble- hen-align”s a egy,
he eads a e i s de-no o assembled and he esul ing
con igs a e hen aligned o he e e ence genome o close
gaps and c ea e sca olds [41]. In ou e e ence guided
assembly pipeline (Figu e 2) we use a combina ion o
hese wo app oaches by mapping bo h ma e-pai ed eads
and de-no o assembled con igs o he e e ence genome
and combining hese alignmen s o p oduce he inal sca -
olds. This app oach has some simila i ies o he p e iously
published “ e e ence-assis ed ch omosome assembly”[42].
Impo an ly, we also demons a e he u ili y o he SOLiD
sequencing echnology (Applied Biosys ems) o whole gen-
ome de-no o sequencing. Due o he sho eads p oduced
by his me hod compa ed o mo e widely used 454 (Roche)
and HiSeq (Illumina) sequence s, he SOLiD pla o m has
no been used be o e o sequencing o e eb a e sized
genomes in a non-model o ganism. Bac e ial [43] and un-
gal [44] genomes ha e, howe e , p e iously been sequenced
solely based on his echnology. E en hough he de-no o
assembly o ou sho ead da a was agmen ed (due o he
sho ead leng hs) we we e s ill able o success ully co e a
la ge p opo ion o he genome using ou e e ence guided
app oach. We used he d a genome bo h o iden i y a as
numbe o SNPs and o pe o m compa a i e genomic
analyses.
Resul s and discussion
SOLiD sequencing
The aw sequencing da a was comp ised o 793 M eads
wi h a ead leng h o 75 bp which we e gene a ed o
he single-end lib a y, 1642 M eads wi h ead leng h o
60 bp × 60 bp which we e gene a ed o he 2 Kb ma e-
pai ed lib a y, and 1548 M eads wi h ead leng h o
60 bp × 60 bp which we e gene a ed o he 5 Kb ma e-
pai ed lib a y. The aw eads a e deposi ed in he NCBI
sequence ead a chi e (SRA) unde he accession numbe
SRA061602. A e quali y and leng h il e ing, 423 M
eads (53.3%) we e e ained o he single-end lib a y,
320 M (75.7%) o which we e 75 bp in leng h. Fo he
2 Kb ma e-pai ed lib a y, 857 M eads (52.2%) we e
e ained a e il e ing, and 663 M (77.4%) o hem we e
60 bp in leng h. Fo hose il e ed eads, 519 M (31.6%)
we e p ope ly pai ed, and he es we e only e ained as
unpai ed eads. Fo he 5 Kb ma e-pai ed lib a y, 847 M
eads (54.7%) we e e ained a e il e ing, 648 M (76.5%)
o which we e o 60 bp in leng h. Fo hose il e ed eads,
520 M (33.6%) we e p ope ly pai ed, and he es we e
only e ained as unpai ed eads. The e o e, 2127 M
high quali y sequencing eads wi h he o al leng h o
Figu e 1 Male black g ouse displaying a a lek.
Wang e al. BMC Genomics 2014, 15:180 Page 2 o 13
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app oxima ely 133 Gb we e kep in downs eam analysis.
I weassume ha hegenomesizeo blackg ouseissimila
o ha o chicken (1.05 G), he es ima ed mean sequencing
co e age o he black g ouse genome was 127X.
Re e ence guided assembly
The e e ence guided assembly is comp ised o se e al
s eps, including de no o assembly, e e ence mapping
and he me ging o hese esul s (Figu e 2). In he i s
s ep, all he 2127 M il e ed high quali y eads we e de
no o assembled by SOAPdeno o. We we e able o gen-
e a e 1298366 p elimina y con igs wi h a o al leng h
o 937 Mb. As expec ed, he de no o assembly was
mo e agmen ed compa ed o some o he s udies which
also used sho - ead sequencing echnologies [15,16,22],
his is because in his s udy we only had h ee sequen-
cing lib a ies wi h a maximum inse size o 5 Kbp and
he sequencing eads p oduced by he SOLiD echnol-
ogy we e ela i ely sho . The SOLiD pla o m is be-
lie ed o p oduce high quali y eads [45]. All he il e ed
da a we used in ou analyses had an e o a e no la ge
han 0.1%. Howe e , he sho ead leng h se iously
a ec s i s pe o mance in pu e de no o assembly. Longe
sequencing eads p oduced by pla o ms such as 454,
ion- o en o PacBio usually p oduce la ge con igs and
such da a could be used o imp o e ou assembly in he
u u e.
In he nex s ep, we aligned all long con igs o he
chicken genome (Figu e 2) and we e able o map 277501
o hem. The o al mapped leng h was 438 Mb. A he
same ime, we also aligned he il e ed and p ope ly pai ed
eads om he ma e-pai ed lib a ies o he chicken gen-
ome esul ing in 451 M success ully mapped eads. These
wo se s o mapped eads we e me ged and his esul ed
in a 805 Mb black g ouse genome backbone sca old.
Finally, we mapped he de no o assembled con igs back
o he black g ouse backbone sca olds and had 1175021
o hem mapped. The e o e, we succeeded o co e 833 Mb
(79.6%) o he 1046 Mb chicken genome, and 4572 o
he 15932 chicken sca olds ( e sion galGal4). We co e ed
826 Mb (82.5%) o he 1001 Mb main chicken ch o-
mosomes (ch omosomes 1-18, and ch omosome Z). In
addi ion, we also e ained 41098 unmapped con igs (a e
disca ding 265 con igs as likely con amina ions) wi h a
o al leng h o 16.6 Mb.
The esul ing black g ouse d a genome assembly
consis ed o 4572 sca olds wi h a o al leng h o 1022 Mb
(o which 833 Mb is sequenced and he es ep esen gaps
in he sequence). The genome assembly is deposi ed in
he NCBI whole genome sho gun (WGS) da abase unde
Figu e 2 Flow cha o ou e e ence guided genome assembly pipeline. All eads we e i s de-no o assembled. In he second s ep bo h
long con igs and o iginal ma e-pai ed eads we e mapped o he chicken e e ence genome and me ged o p oduce he black g ouse backbone
sca olds. Finally gap- illing was pe o med by mapping all con igs (o a leas 100 bp) back o he backbone sca olds, p oducing he d a genome
assembly. In addi ion all con igs (a leas 200 bp long) no mapping o he backbone sca olds we e added o he assembly a e emo ing sequences
a ising om possible con amina ion using a BLAST app oach. Fo mo e de ails abou he p ocedu es please see he Me hods sec ion. In he igu es o
he igh he chicken e e ence genome is indica ed by he blue line while eads, con igs and sca olds om he black g ouse a e shown in ed. The
ligh ed pa s o he inal sca old indica e egions wi h gaps (Ns) in he black g ouse sequence. Wi hin b acke s in he boxes o he le a e he
so wa e used o each di e en s age o he assembly p ocess.
Wang e al. BMC Genomics 2014, 15:180 Page 3 o 13
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he submission numbe JDSL00000000. Among he sca -
olds, he 29 la ges , co esponding o he chicken ch o-
mosomes 1 ~ 28 and ch omosome Z, had a o al leng h
o 1001 Mb (826 Mb sequenced). The a e age co e age
(p opo ion o he si es sequenced) o he 29 ch omosomes
was 81.5%. Howe e , his co e age was no dis ibu ed
e enly ac oss he ch omosome sca olds o ac oss he
ch omosomal egions (Figu e 3, Addi ional ile 1). We no-
iced ha ch omosome 16, ch omosome 25, ch omosome
27 and ch omosome Z we e no well co e ed. Ch omo-
some Z is he a ian sex ch omosome and ch omosome 16
ha bou s he MHC genes [35,46]. These migh be mo e
di e gen be ween black g ouse and chicken han he es
o he genome, which may ha e led o he poo assembly.
Fo ch omosome 16, an addi ional eason migh be ha
he chicken assembly o his ch omosome is s ill no
pe ec and con ain la ge N chunks. Fu he mo e, we
ha e p e iously shown ha when compa ing his egion
among di e en galli o m species, he e a e se e al gene
copy di e gences as well as genomic in e sions in he
MHC egion on ch omosome 16 [40]. We u he exam-
ined he quali y o he black g ouse d a genome, and
20 (%)
100
80
60
40
100 200 (Mb)
0
10 20 (Mb)
0
ch 1
ch 2
ch 3
ch 4
ch 5
ch Z
4 8 (Mb)
0
ch 6
ch 7
ch 8
ch 9
ch 10
ch 11
ch 12
ch 13
ch 14
ch 15
ch 17
ch 18
ch 19
ch 20
Sliding window size 1Mb
Sliding window size 100Kb
Sliding window size 50Kb
ch 16
ch 21
ch 22
ch 23
ch 24
ch 25
ch 26
ch 27
ch 28
Figu e 3 Hea map showing he p opo ion o he egions sequenced on he ch omosome sca olds.
Wang e al. BMC Genomics 2014, 15:180 Page 4 o 13
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ound ha al hough he sequence co e age was high, he
sca old sequences we e highly agmen ed (Figu e 4).
I he d a genome is spli a all ‘N’si es p esen on he
sca olds, i has 3071478 con inuous sequenced blocks.
This is no unexpec ed since he SOLiD da a was sho
andweuseda e e enceguidedapp oach op oduce
he d a genome, and he exis ing bioin o ma ic ools
a ailable a e no ma u e enough in dealing wi h his s a -
egy. The SAM ools pipeline we used gene a es consensus
sequences solely based on he coo dina es o he e e ence
genome, which migh in oduce a numbe o addi ional ‘N’s
in he esul ed sequences. In addi ion, a numbe o he long
‘N’chunks a e also p esen in he e e ence chicken
genome, and migh he eby be in oduced in o he black
g ouse d a genome h ough he e e ence guided assem-
bly p ocess.
Anno a ion
The agmen ed s a e o he d a genome limi ed ou
abili y o sys ema ically pe o m ab-ini io p edic ions o
genes o genomic epea s. Ins ead we used compa a i e
me hods o iden i y he gene egions and he genomic
epe i i e egions. F om he ecip ocal BLAST esul , we
ound ha 14826 chicken genes had homologs on he
black g ouse genome (Table 1, Addi ional ile 2). The
coding egions o hose homolog genes co e ed abou
45.4 Mb o he black g ouse d a genome. We also
checked how well each coding sequences o he chicken
genes we e co e ed, as his could in e he comple eness
o he anno a ed genes o he black g ouse genome
(Figu e 5). We ound 5592 genes, wi h a g ea e han
90% co e age o he coding egions. This is, howe e ,
only a ough es ima e, as he leng h o coding sequences
could a y be ween black g ouse genes and chicken genes.
We also no iced he in e es ing ‘U’shape o he plo , ha
is, he coding egions o a la ge majo i y o he genes a e
ei he co e ed o a e y la ge ex en (genes wi h co e age
abo e 80%) o o a e y small ex en (genes wi h co e age
less han 20%). This may be explained i some genes ha
a e highly di e gen be ween black g ouse and chicken
could no be p ope ly aligned in he e e ence guided
assembly s ep.
Looking a he dis ibu ion o he anno a ed genes ac oss
he sca olds, we ound ha he majo i y o he genes we e
iden i ied on he 29 main ch omosome sca olds. In e es -
ingly, 634 genes we e iden i ied om he unmapped
con igs, sugges ing ha hose genes could be no included
in he e e ence chicken genome, o be highly di e gen
be ween black g ouse and chicken. The a e age gene
densi y o he 29 main ch omosome sca olds was 1.41E-5
gene/nucleo ide. Ch omosome 1 had he highes numbe
o genes (2017) as i was he longes ch omosome.
Ch omosome 16 had he highes gene densi y o 1.03E-4
gene/nucleo ide, while ch omosome Z had he lowes gene
densi y o 7.99E-6 gene/nucleo ide.
Genomic epea s we e iden i ied using all known a ian
genomic epea s as e e ences. We ound a o al leng h
o 64.7 Mb genomic epea s, which accoun ed o 6.34%
o he d a genome (Table 2). These numbe s a e lowe
han hose o chicken and zeb a inch, bu simila o
hose o u key [35,47,48]. The chicken genome and he
zeb a inch genome we e de eloped exclusi ely by Sange
Numbe o sequence-blocks/ To al leng h (bp)
Numbe o ‘N’ blocks/ To al leng h (bp)
Leng h o sequence-block (bp) Leng h o ‘N’ block (bp)
Figu e 4 Dis ibu ions o he con inuous sequence-blocks and he in e lea ed ‘N’blocks. The blue lines indica e he o al leng h (bp)
o he espec i e blocks. The sequenced blocks la ge han 10000 bp and he ‘N’blocks la ge han 30000 bp we e binned a he end o
he axis.
Table 1 Numbe o genes om o he bi d genomes ound
o be homologous o he black g ouse d a genome
Chicken Tu key Zeb a inch
Numbe o genes 17934 15006 18618
Black g ouse homologs 14826 13721 12573
Pe cen age (%) 82.7 91.4 67.5
Wang e al. BMC Genomics 2014, 15:180 Page 5 o 13
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sequencing and ha e a be e quali y han ha o u key
and black g ouse. This could be he eason why we ound
less genomic epea s in he black g ouse d a genome.
The black g ouse d a genome also had low numbe s
o simple epea elemen s and low complexi y egions
compa ed o he o he species. Howe e , his can be
explained by he agmen ed na u e o he black g ouse
d a genome whe e simple epea s elemen s and low
complexi y egions may simply be ep esen ed by ‘N’
blocks, p e en ing he p og am om de ec ing hem.
Iden i ica ion o SNPs
E en hough he genome sequence is only based on one
indi idual, he ac ha his wild, ou b ed bi d was highly
he e ozygous allowed us o iden i y a la ge numbe o
SNPs [15]. To his end, we mapped all he il e ed se-
quencing eads o he black g ouse d a genome. 983 M
eads, including he ones om he single-end lib a y,
he single ons om he ma e-pai ed lib a ies and he pai ed
eads o he wo ma e-pai ed lib a ies, we e mapped o
he genome wi h a e age co e age o 57.2X. We se he
co e age cu -o o he SNP calling as 50 o 100X. We
only accep ed SNP si es wi h ce ain le el o co e age o
ensu e he quali y o he SNP calling bu also o a oid he
si es wi h unusually high co e age, as hey migh be
he esul o inco ec mapping o eads om duplica ed
egions. Thus, we inally ob ained 964054 high quali y
SNPs, 31993 (3.3%) o which we e om he coding e-
gions(Table3).The ansi ion/ ans e sion a ioo he
SNPs was 2.05. The SNP densi y o his black g ouse
indi idual was 0.114%, which was highe han ha o
u key (0.064%) [48]. Howe e , he indi idual used in
he u key genome sequencing p ojec was inb ed, whe eas
he black g ouse indi idual we used was om a la ge ou -
b ed na u al popula ion. In e es ingly, we ound ha he
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0
1000
2000
3000
4000
5000
6000
P o
p
o ion o he codin
g
e
g
ion co e ed
Numbe o genes
Figu e 5 His og am o he p opo ion o he coding egions co e ed o he anno a ed black g ouse genes.
Table 2 In o ma ion on epea elemen s iden i ied om
sequenced bi d genomes
Black
g ouse
Chicken Tu key Zeb a
inch
To al leng h o epea s (Mb) 64.7 111.2 60.9 97.5
Pe cen age in genome (%) 6.34 10.63 5.74 7.91
Numbe o speci ic elemen s
SINEs 4869 5207 4745 5512
LINEs 151426 183599 140717 133331
L2/CR1/Rex 151298 183464 140575 126284
LTR elemen s 17792 32080 6325 78514
Re o i al 17733 32015 6266 78185
DNA ansposons 19300 21971 3919 4391
Small RNA 1440 1806 1609 1897
Sa elli es 1433 3816 1247 488
Simple epea s 50145 133371 83093 123377
Low complexi y 65102 143762 125682 196866
Unclassi ied 2017 2222 2028 2334
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SNP densi y o black g ouse was close o ha o he gian
panda (0.135% o au osomes), which was in e ed om a
cap i e bu ou b ed indi idual [15].
We u he in es iga ed he SNPs on he 29 la ge
ch omosome sca olds (Table 3, Addi ional ile 1). We
classi ied he ch omosomes in o ou ca ego ies: mac o-
ch omosomes (ch omosome 1 ~ 5), in e media e-ch o-
mosomes (ch omosome 6 ~ 10), mic o-ch omosomes
(ch omosome 11 ~ 28) and sex ch omosome (ch omo-
some Z). We ound ha he mac o-ch omosomes had
he highes he e ozygosi y while he sex ch omosome had
he lowes . The he e ozygosi y o he mic o-ch omosomes
was also low. This migh be because ha he mic o-
ch omosomes ha e a highe gene densi y in he black
g ouse. In con as , he sex ch omosome had he lowes
densi y o genes bu also had a low he e ozygosi y. Simila
pa e ns ha e been obse ed in a wide a ie y o o ganisms
and a e explained by he ac ha he e ec i e popula ion
size o ch omosome Z is heo e ically 0.75 compa ed
o ha o he au osomes [49]. In addi ion, he educed
a ia ion on he Z (co esponding o X in mammals and
lies) could also be in e p e ed as he esul o as e e olu-
ion and pu i ying selec ion [50-52].
Compa a i e genomics
Since he sca olds o he black g ouse d a genome we e
de eloped by using he chicken genome as e e ence, we
could no in es iga e he genomic a ia ions o black
g ouse, chicken and o he species om a genomic e-
a angemen pe spec i e, howe e , he sequences allowed
us o conduc a comp ehensi e compa a i e genomic ana-
lysis a he le el o nucleo ide a ia ion. Fo his analysis,
we ocused on he main ch omosomes (ch omosome 1-28
and ch omosome Z) and examined he nucleo ide di e -
gence (numbe o a iable si es pe uni ) be ween black
g ouse, chicken and u key. The downloaded chicken
genome was spli o 187307 sequences, o which 181105
(96.7%) could be mapped o he black g ouse main
ch omosome sca olds (ch omosome 1-28 and ch omo-
some Z). This alignmen co e ed 795 M (96.2%) o he
sequenced si es o he main black g ouse ch omosomes.
The downloaded u key genome was spli o 336344
sequences, o which 328727 (97.7%) could be mapped o
he main black g ouse ch omosome sca olds. This align-
men co e ed 703 M (85.1%) o he sequenced si es o he
main black g ouse ch omosomes. The u key genome
had a highe mapping pe cen age bu a much lowe
co e age o he sequenced si es o he black g ouse gen-
ome, as he u key genome sequences we e o lowe qual-
i y (con aining many un esol ed nucleo ides ‘N’,)
compa ed o hose o chicken.
The a e age nucleo ide di e gence be ween he 29 black
g ouse and chicken ch omosomes was 0.099 ± 0.009, wi h
he di e gence be ween black g ouse and u key was
0.101 ± 0.009. Those nucleo ide di e gence es ima es we e
a li le lowe compa ed o he s udies on chicken and
u key [53,54], his, howe e , migh be because we used
a genome mapping app oach o comple e he alignmen ,
which could make us miss he mos highly di e gen se-
quences. The black g ouse, he chicken and he u key
a e closely ela ed species. Coun e o ou indings he e,
phylogene ic analysis sugges s ha he black g ouse is
mo e closely ela ed o u key han chicken [33,46]. This
migh be because, since we used chicken genome as e e -
ence o cons uc he black g ouse d a genome o he
he e ozygous nucleo ide si es, he choice o he nucleo ides
could be biased owa ds he chicken e e ence genome. To
u he in es iga e he nucleo ide di e gence, we g ouped
he ch omosomes in o ou ca ego ies (mac o-ch omo-
some, in e media e-ch omosome, mic o-ch omosome, sex
ch omosome) as desc ibed in he las sec ion. We
ound ha he nucleo ide di e gence o in e media e-
ch omosomes was sligh ly lowe han ha o he
mac o-ch omosomes, and he nucleo ide di e gence o
mic o-ch omosomes was sligh ly highe han ha o
mac o-ch omosomes and in e media e-ch omosomes
(Figu e 6). The nucleo ide di e gence o he Z ch omo-
some was also highe han o he au osomes. The obse -
a ion o inc eased di e gence a es on sex ch omosomes
is o en e e ed o as he as e X e ec [55]. This pa e n
is gene ally hough o a ise om he smalle e ec i e
popula ion size o sex ch omosome compa ed o au o-
somes, o an inc eased accumula ion o ecessi e adap i e
mu a ions [50,52,56,57].
Table 3 Numbe and densi y o single nucleo ide polymo phisms (SNPs) iden i ied in he genome sequence om one
ou b ed black g ouse indi idual
Numbe
o SNP
SNP
densi y (%)
SNP ype
T ansi ion T ans e sion
A/G C/T A/C A/T C/G G/T
To al 949254 0.114 320842 320314 79442 84824 60193 80439
Mac o-ch omosomes (1 ~ 5) 601867 0.120 201789 201258 51505 56799 37937 52579
In e media e-ch omosomes (6 ~ 10) 146269 0.117 49447 49574 12547 12736 9554 12411
Mic o-ch omosomes (11 ~ 28) 161369 0.111 54160 53843 13324 12465 10918 13459
Ch omosome Z 39749 0.071 15446 15639 2066 2824 1784 1990
Wang e al. BMC Genomics 2014, 15:180 Page 7 o 13
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Finally, we calcula ed he nucleo ide di e gence by 50 Kb
sliding window size o sc een o he highly di e gen
genome egions be ween black g ouse and chicken and
be ween black g ouse and u key. A p e ious s udy sug-
ges ed he di e gen a e o he galli o m MHC egion
was app oxima e 0.15 [40]. He e, we used a di e gence
a e o 0.2 as he cu -o and iden i ied 106 egions
which had a high nucleo ide di e gence a e (exampled
in Figu e 7, Addi ional ile 3). These egions could po en-
ially ha bou genes o gene egula o y elemen s which
a e impo an o some speci ic pheno ypic a ibu es
o he black g ouse. Among he iden i ied genomic
egions, 45 con ained genes o gene agmen s and a
o al o 67 genes we e localized in hese high di e gence
egions. Those genes a e impo an o he u u e in-dep h
s udieso helineagespeci ice olu iono heblackg ouse.
Conclusions
In his s udy, using he chicken genome as a e e ence, we
success ully assembled he whole d a genome o black
g ouse. The d a genome consis s o 4572 sca olds
wi h a o al leng h o 1022 Mb (833 Mb sequenced),
and addi ional 41098 unsca oled con igs wi h o al
leng h o 16.6 Mb. This co esponds o a high co e age
o he chicken ch omosomes 1 ~ 28 and ch omosome Z,
wi h a o al leng h o 1001 Mb (826 Mb sequenced). Al-
hough he con inuously sequenced blocks on he sca olds
a e agmen ed, he d a genome has a good co e age
o he homologous chicken genes, and 14826 (82.7%) o
he chicken genes we e iden i ied on he black g ouse
d a genome. No ably, 33.0% o he coding egions o
he homologous genes ha e mo e han 90% p opo ion
o hei sequences co e ed. To ou knowledge, his is
he i s imeala geeuka yo egenomewasde eloped
by SOLiD sho sequencing echnology and e e ence
guided assembly bioin o ma ic pipeline. Ou s udy demon-
s a es how a mode a e sequencing e o can be combined
wi h exis ing genome e e ences o accomplish a la ge
genome p ojec . We iden i ied a la ge numbe (949254)
o SNPs and iden i ied he genomic egions we sugges
a e impo an o he lineage speci ic e olu ion o black
g ouse. F om he abo e analysis, we no e ha he
sex ch omosome (ch omosome Z) had lowe e e ence
assembly e iciency, lowe SNP densi y bu a highe
nucleo ide di e gence be ween black g ouse and o he
galli o m species. Those mul iple e idences suppo
he as e X (Z) hypo hesis o he sex ch omosome, which
s a es ha he ch omosome X (Z) e ol es as e han he
au osomes due o i s lowe e ec i e popula ion size and
ecombina ion a e. We also obse ed ha mic och omo-
some 16 which ha bou s he MHC egion in galli o ms
was highly di e gen among species which may indica e
as e e olu ion in his genomic egion.
Me hods
DNA sampling, ex ac ion and sequencing
The black g ouse indi idual used in his s udy was a male
collec ed by a licensed hun e in he win e hun ing
Mac o
ch omosome
In e media e
ch omosome
Mic o
ch omosome
Sex
ch omosome
CK TK CK TK CK TK CK TK
Nucleo ide di e gence
Figu e 6 Nucleo ide di e gence o he ch omosome g oups. Mac o-ch omosome: ch omosome 1 ~ 5. In e media e-ch omosome:
ch omosome 6 ~ 10. Mic o-ch omosome: ch omosome 11 ~ 28. Sex ch omosome: ch omosome Z. CK ep esen s he compa ison be ween
black g ouse and chicken. TK ep esen s he compa ison be ween black g ouse and u key.
Wang e al. BMC Genomics 2014, 15:180 Page 8 o 13
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