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 .
De no o ansc ip ome assembly and i s anno a ion o he aposema ic wood ige
mo h (Pa asemia plan aginis)
Gala za, Juan; Kisho , Dhaygude; Mappes, Johanna
Gala za, J., Kisho , D., & Mappes, J. (2017). De no o ansc ip ome assembly and i s
anno a ion o he aposema ic wood ige mo h (Pa asemia plan aginis). Genomics
Da a, 12, 71-73. h ps://doi.o g/10.1016/j.gda a.2017.03.008
2017
De no o ansc ip ome assembly and i s anno a ion o he aposema ic
wood ige mo h (Pa asemia plan aginis)
Gala zaJuan A.
a,
⁎
,1
, Kisho Dhaygude
b,1
, Johanna Mappes
a
a
Cen e o Excellence in Biological In e ac ions, Dep . o Biological and En i onmen al Sciences, Uni e si y o Jy äskylä, Finland
b
Cen e o Excellence in Biological In e ac ions, Uni e si y o Helsinki, Finland
abs ac a icle in o
A icle his o y:
Recei ed 6 Ma ch 2017
Accep ed 19 Ma ch 2017
A ailable online 21 Ma ch 2017
In his pape we epo he public a ailabili y o ansc ip ome esou ces o he aposema ic wood ige mo h
(Pa asemia plan aginis). A comp ehensi e assembly me hods, quali y s a is ics, and anno a ion a e p o ided.
This e e ence ansc ip ome may se e as a use ul esou ce o in es iga ing unc ional gene ac i i y in apose-
ma ic Lepidop e an species. All da a is eely a ailable a he Eu opean Nucleo ide A chi e (h p://www.ebi.ac.
uk/ena) unde s udy accession numbe : PRJEB14172.
© 2017 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license (h p://
c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. Di ec link o deposi ed da a
h p://www.ebi.ac.uk/ena. S udy accession numbe : PRJEB1417.
2. Specifica ions
O ganism/cell line/ issue Wood ige mo h (Pa asemia plan aginis)/whole la a
Sex Unde e mined
Sequence o a ay ype Illumina HiScanSQ
Da a o ma FASTQ
Expe imen al ac o s De no o assembly, comple eness assessmen ,
and anno a ion
Expe imen al ea u es RNA-seq om whole la ae (n= 16) o
Wood ige mo h (Pa asemia plan aginis)
om di e en de elopmen al s ages.
Consen N/A
Sample sou ce loca ion Jy äskylä, Cen al Finland
3. In oduc ion
Many plan s and animals ad e ise unpala abili y h ough conspicu-
ous colou a ion as a o m o wa ning signals o po en ial p eda o s (i.e.
aposema ism). This de ensi e s a egy is used by many Lepidop e ans
(bu e flies and mo hs), and while i s ecological and e olu iona y con-
sequences a e ela i ely well-s udied [2,14] sca ce in o ma ion is a ail-
able abou hei molecula bases. The wood ige mo h (Pa asemia
plan aginis) is a diu nal aposema ic species ha shows conside able col-
ou a ia ion h oughou i s dis ibu ion ange [6]. I has been ecen ly
classified as A c ia plan aginis [13]. Two male colou mo phs and one e-
male colou mo ph co-exis wi hin local popula ions. I has been shown
ha he wo male colou mo phs di e in hei wa ning signal e ficacy,
one being be e p o ec ed han he o he agains a ian p eda o s [9].
Fu he mo e, in sou he n Finland, he gene ic composi ion o he popu-
la ions fluc ua es be ween gene a ions [4].Thus, hisspeciesp o idesa
aluable oppo uni y o in es iga e equency-dependen selec ion p o-
cesses in na u e. Howe e , he wa ning signals displayed by adul s a e
p e-de e mined du ing he la al s age, when bodily esou ces a e allo-
ca ed o de e mine hei shape and pa e n. A e me amo phosing in o
he adul s age, no u he de elopmen o adap a ions ake place a he
pheno ypic le el. Thus, unc ional gene ac i i y du ing ea ly li e-s ages
mus be in es iga ed o gain insigh abou i s possible e ec s on he
adul pheno ype.
He e we epo a de no o ansc ip ome assembly o he wood ige
mo h a i s la al s age. Ou aim was o ob ain a high-co e age, high-
quali y e e ence ansc ip ome ep esen a i e o di e en de elop-
men al s ages. The da a p esen ed he e a e he fi s ansc ip ome e-
sou ces a ailable o he wood ige mo h.
4. Expe imen al design, ma e ials and me hods
La ae o igina ed om popula ions o Cen al Finland. A o al o 16
la ae om ins a 1 o ins a 5 we e selec ed o sequencing. All la ae
we e ed dandelion (Ta axacum spp.) and ea ed indi idually in pe i
dishes be o e imme sion in RNAla e s abilising bu e . All samples
we e kep a −20C° un il RNA ex ac ion.
Genomics Da a 12 (2017) 71–73
⁎Co esponding au ho .
E-mail add ess: juan.gala za@jyu.fi(J.A. Gala za).
1
These au ho s con ibu ed equally o he ealiza ion o his pape .
h p://dx.doi.o g/10.1016/j.gda a.2017.03.008
2213-5960/© 2017 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Con en s lis s a ailable a ScienceDi ec
Genomics Da a
jou nal homepage: www.else ie .com/loca e/gda a
4.1. RNA ex ac ion
To al RNA was ex ac ed using RNeasy Mini Ki (Qiagen) acco ding
o manu ac u e 's ins uc ions wi h addi ional T iReagen (MRC, Inc.)
and DNase (Qiagen, Valencia, US.A.), ea men s. The quali y and quan-
i y o o al RNA was inspec ed in a BioAnalyze 2100 using RNA 6000
Nano Ki (Agilen ). Subsequen ly, mRNA was isola ed by means o wo
isola ion cycles using Dynabeads mRNA pu ifica ion ki (Ambion®)
and quan ified using RNA 6000 Pico Ki in a BioAnalyze 2100 (Agilen ).
Pai -end (2 × 100 pb) cDNA lib a ies we e cons uc ed o each sample
acco ding o Illumina's T uSeq S anded HT p o ocol. The lib a ies we e
indi idually indexed and sequenced in an Illumina HiScanSQ sequence
a he DNA sequencing and genomics labo a o y, Ins i u e o Bio echnol-
ogy, o he Uni e si y o Helsinki, Finland.
4.2. T ansc ip ome assembly
The quali y o he aw eads was fi s inspec ed wi h Fas QC (h p://
www.bioin o ma ics.bab aham.ac.uk/p o-jec s/ as qc/). Based on his
ini ial quali y check, we used he FASTX oolki (h p://hannonlab.cshl.
edu/ as x_ oolki /) o emo e low quali y bases and sequencing a i-
ac s. Bases wi h a Ph ed quali y sco e o b25 we e fil e ou , and
eads sho e han 85 bases a e imming we e emo ed. Pai -end
eads we e hen so ed and synch onized using cus om sc ip s.
We used he high-quali y eads om all samples ob ained a e
Fas QC and FASTX o pe o m an ini ial assembly (K25_Assembly)
using T ini y ( ini y naseq_ 2013-02-25) so wa e [5] wi h he ollow-
ing pa ame e s: 4 CPUs o Inchwo m and Bu e fly, a maximum mem-
o y 200 GB, a minimum con ig leng h o 200 bp, and K-me = 25. The
de aul K-me o 25 eco e ed mos ull-leng h ansc ip s ac oss a
b oad ange o exp ession le els. To iden i y any unassembled eads,
we mapped back he eads o he K25_Assembly using bow ie .
0.12.7 [8]. The unassembled eads we e hen used o cons uc wo u -
he assemblies namely; K21_Assembly and K29_Assembly using wo
di e en K-me se ings o 21 and 29 espec i ely. A ou h assembly
Table 1
P ope ies and s a is ic o he Final_Assembly ansc ip ome o he wood ige mo h
(Pa asemia plan aginis).
To al unigenes 54,657
Unigenes a e ibosomal fil e ing 54,346
N50(bp) 10,747
Mean co e age 39.12×
No. mapped eads 366,046,742(98.44%)
Anno a ed in n 17,800
Anno a ed in Swiss-P o 6309
Anno a ed in GO 16,936
Anno a ed in In e -P o 20,020
Fig. 1. (A) O holog hi a ios (OHR) o unigenes om Final_Assembly agains silkwo m (Bobyx mo i) genome. (B) Co e age ob ained om he di e en assemblies wi h a ying K-me
leng h om 21 o 29.
72 J.A. Gala za e al. / Genomics Da a 12 (2017) 71–73
(Final_Assembly) was cons uc ed by combining he K25_Assembly
wi h he o he wo K21 and K25 assemblies using CAP3 [7]. The Sca -
olding so wa e was un wi h de aul pa ame e s making su e ha
he minimum o e lap be ween wo con igs was a leas 100 bp wi h a
95% sequence simila i y o building supe con igs.
We mapped back all eads o he Final_Assembly using bow ie as
abo e o calcula e he o e all exp ession p ofile o all ansc ip s. We
hen emo ed any miss-assemblies o assembly e o s by manual in-
spec ion o ansc ip s ha showed RPKM (Reads Pe Kilobase o an-
sc ip pe million mapped eads) alues o b1. Finally, we used
Vma ch (h p://www. ma ch.de) ofil e ou possible edundan
unigenes p esen in his Final_Assembly. The comple e assembly
wo kflow is p esen ed in Supplemen a y file 1.
4.3. T ansc ip ome alida ion
T ansc ip ome alida ion o non-model o ganisms is especially
challenging because o he noisy na u e o ansc ip s and lack o a e -
e ence genome o pe o m a guided assembly. O hology check wi h
he genome o he closes e e ence species a ailable is one way o as-
sess he quali y o he de no o assembly. Hence, we compu ed he
o holog hi a io [11] o he Final_Assembly using he silkwo m mo h
(Bombyx mo i; axa ID: 17,701 Uni e 90), which is he closes species
wi h a ho oughly anno a ed genome. Ra ios close o 1 a e indica i e
ha con igs o ansc ip s (i.e. unigene) ma ching he o holog locus
ha e been ully assembled.
4.4. T ansc ip ome anno a ion
Fo anno a ing he ansc ip ome, he Final_Assembly was fi s
checked o possible ibosomal con amina ion ha may ha e su i ed
he RNA pu ifica ion phase. The ansc ip ome was blas ed [1] (BLASTn;
e- alue ≤10
−5
) agains publicly a ailable ibosomal da abases o a -
chaea, bac e ia, and euka yo e domains
(SILVA_123_SSUPa c_Taxa_T unc & SILVA_123_LSUPa c_Taxa_T unc -
Release 123; [12]. All unigenes ha showed significan hi s we e e-
mo ed (b3%). The emaining unigenes we e compa ed agains a non-
edundan p o ein da abase (n ) (NCBI; las upda ed 30-05-2016) and
he Swiss-P o (las upda ed 26–05-2016) o e ie e basic anno a ion
using BLASTx. A e blas ing, all hi s ha showed b70% amino acid iden-
i y, sequence leng h o b200 bp, and e- alue ≤10
−5
we e fil e ed ou
using cus om sc ip s. Gene on ology e ms (GO) and in o ma ion o
he p o ein amily was ob ained using Blas 2Go .4.0 [3].
5. Resul s
A o al o 372,037,058 pai s o eads we e ob ained om he se-
quencing uns (Ph ed +33; ASCII ange “!” o “J”). A e imming and
fil e ing, 371,847,565 pai s o eads (%GC 45) wi h a leng h o 85 bp
we e used o he ansc ip ome assemblies. The basic desc ip o s and
quali y me ics o he Final_Assembly a e p esen ed in Table 1 and Fig.
1. To e alua e he bes ansc ip assembly me hod (TA), we compa ed
he comple eness (co e age) o TA's p oduced om each K-me assem-
bly o he closes anno a ed genome (Bombyx mo i), as p oposed by
[10]. The esul s showed ha me ging assemblies o di e en K-me s
yield he highes co e age (Fig. 1).
5.1. Anno a ion esul s
A o al o 17,800 unigenes e u ned a blas hi wi h e- alue ≤10
−5
and b70% amino acid iden i y. O hese unigenes, 16,036 had gene on-
ology (GO) anno a ion a ailable wi h a mean GO le el o 6.2 ac oss bi-
ological p ocesses (P), molecula (F) unc ion and cellula componen s
(C) ca ego ies. The main P,F,C a e emo ing edundan GO e ms a e
summa ized in Supplemen a y file 1. The numbe o unigenes anno a -
ed o he di e en da abases is shown in Table 1 and i s unc ional anno-
a ion is p o ided in he Supplemen a y file 2.
Acknowledgemen s
This p ojec was unded by he Cen e o Excellence in Biological In-
e ac ion, ia he Academy o Finland (P ojec No.252411). The au ho s
a e hank ul o he Finish Cen e o Science (CSC-IT) o access o com-
pu a ional esou ces, and o Pe i Au inen and La s Paulin om he
DNA sequencing and genomics labo a o y, Ins i u e o Bio echnology,
o he Uni e si y o Helsinki, Finland.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h p://dx.
doi.o g/10.1016/j.gda a.2017.03.008.
Re e ences
[1] S.F. Al schul, W. Gish, W. Mille , E.W. Mye s, D.J. Lipman, Basic local alignmen
sea ch ool. J. Mol. Biol. 215 (1990) 403–410.
[2] C.A. Ba ne , M. Ba eson, C. Rowe, S a e-dependen decision making: educa ed p ed-
a o s s a egically ade o he cos s and benefi s o consuming aposema ic p ey.
Beha . Ecol. 18 (2007) 645–651.
[3] A. Conesa, S. Go z, J.M. Ga cia-Gomez, e al., Blas 2GO: a uni e sal ool o anno a-
ion, isualiza ion and analysis in unc ional genomics esea ch. Bioin o ma ics 21
(2005) 3674–3676.
[4] J.A. Gala za, O. Nokelainen, R. Ash afi, R.H. Hegna, J. Mappes, Tempo al ela ionship
be ween gene ic and wa ning signal a ia ion in he aposema ic wood ige mo h
(Pa asemia plan aginis). Mol. Ecol. 23 (2014) 4939–4957.
[5] M.G. G abhe , B.J. Haas, M. Yassou , e al., Full-leng h ansc ip ome assembly om
RNA-Seq da a wi hou a e e ence genome. Na . Bio echnol. 29 (2011) 644–652.
[6] R.H. Hegna, J.A. Gala za, J. Mappes, Global phylogeog aphy and geog aphical a ia-
ion in wa ning colo a ion o he wood ige mo h (Pa asemia plan aginis). J.
Biogeog . 42 (2015) 1469–1481.
[7] X. Huang, A. Madan, CAP3: a DNA sequence assembly p og am. Genome Res. 9
(1999) 868–877.
[8] B. Langmead, C. T apnell, M. Pop, S.L. Salzbe g, Ul a as and memo y-e ficien align-
men o sho DNA sequences o he human genome. Genome Biol. 10 (2009) R25.
[9] O. Nokelainen, R.H. Hegna, J.H. Reudle , C. Linds ed , J. Mappes, T ade-o be ween
wa ning signal e ficacy and ma ing success in he wood ige mo h. P oc. R. Soc. B
Biol. Sci. 279 (2012) 257–265.
[10] S. O'Neil, S. Em ich, Assessing de no o ansc ip ome assembly me ics o consis-
ency and u ili y. BMC Genomics 14 (2013) 465.
[11] S.T. O'Neil, J.D.K. Dzu isin, R.D. Ca michael, e al., Popula ion-le el ansc ip ome se-
quencing o nonmodel o ganisms E ynnis p ope ius and Papilio zelicaon. BMC Geno-
mics 11 (2010) 310.
[12] C. Quas , E. P uesse, P. Yilmaz, e al., The SILVA ibosomal RNA gene da abase p o-
jec : imp o ed da a p ocessing and web-based ools. Nucleic Acids Res. 41 (2013)
D590–D596.
[13] K. Rönkä, J. Mappes, L. Kaila, N. Wahlbe g, Pu ing Pa asemia in i s phylogene ic
place: a molecula analysis o he sub ibe A c iina (Lepidop e a). Sys . En omol.
41 (2016) 844–853.
[14] P.J. Weldon, G.M. Bu gha d , E ol ing dé en e: he o igin o wa ning signals ia con-
cu en ecip ocal selec ion. Biol. J. Linn. Soc. 116 (2015) 239–246.
73J.A. Gala za e al. / Genomics Da a 12 (2017) 71–73