Whole genome sequencing of the black grouse (Tetrao tetrix): reference guided assembly suggests faster-Z and MHC evolution

This is an elec onic ep in o he o iginal a icle. This ep in may di e om he o iginal in pagina ion and ypog aphic de ail. Au ho (s): Ti le: Yea : Ve sion: Please ci e he o iginal e sion: All ma e ial supplied ia JYX is p o ec ed by copy igh and o he in ellec ual p ope y igh s, and duplica ion o sale o all o pa o any o he eposi o y collec ions is no pe mi ed, excep ha ma e ial may be duplica ed by you o you esea ch use o educa ional pu poses in elec onic o p in o m. You mus ob ain pe mission o any o he use. Elec onic o p in copies may no be o e ed, whe he o sale o o he wise o anyone who is no an au ho ised use . 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 h p://www.biomedcen al.com/1471-2164/15/180 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 h p://www.biomedcen al.com/1471-2164/15/180 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 h p://www.biomedcen al.com/1471-2164/15/180 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 h p://www.biomedcen al.com/1471-2164/15/180 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 Wang e al. BMC Genomics 2014, 15:180 Page 6 o 13 h p://www.biomedcen al.com/1471-2164/15/180 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 h p://www.biomedcen al.com/1471-2164/15/180 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 h p://www.biomedcen al.com/1471-2164/15/180