DATA NOTE
ERGA-BGE e e ence genome o Hanak's ba (Pipis ellus
hanaki), an IUCN Vulne able species es ic ed o o es -like
bio opes
[ e sion 2; pee e iew: 2 app o ed]
Panagio is Geo gakakis 1, Danae Ka akasi1,2, Pe os Lymbe akis 1,
Manolis Papadimi akis1, Manos S a akis 1,2, Ele he ios Bi zilekis 1,
Nikolaos Poulakakis1-3, As id Böhne 4, Ri a Mon ei o 4, Rosa Fe nández5,
Nu ia Escude o5, Genoscope Sequencing Team, Alice Moussy6, Co inne C uaud6,
Ka ine Labadie6, Lola Demi djian7, Sophie Mangeno 7, Ca oline Belse 7,
Pa ick Wincke 7, Ped o H. Oli ei a 7, Jean-Ma c Au y7, Leanne Hagge y 8,
Swa i Sinha 8, Fe gal Ma in8, Chia a Bo oluzzi 9
1Na u al His o y Museum o C e e, School o Sciences and Enginee ing, Knossos A enue, Uni e si y o C e e, He aklion, GR-71409,
G eece
2Depa men o Biology, School o Sciences and Enginee ing, Vassilika Vou on, Uni e si y o C e e, He aklion, GR-70013, G eece
3Founda ion o Resea ch and Technology – Hellas (FORTH), Ins i u e o Molecula Biology and Bio echnology (IMBB), He aklion, GR-
70013, G eece
4Leibniz Ins i u e o he Analysis o Biodi e si y Change, Museum Koenig Bonn, Adenaue allee 127, Bonn, 53113, Ge many
5Me azoa Phylogenomics Lab, Passeig ma í im de la Ba celone a 37-49., Ins i u e o E olu iona y Biology (CSIC-UPF), Ba celona,
08003, Spain
6Genoscope, Ins i u F ançois Jacob, CEA, CNRS, Uni E y, Uni e si é Pa is-Saclay, E y, 91057, F ance
7Génomique Mé abolique, Genoscope, Ins i u F ançois Jacob, CEA, CNRS, Uni E y, Uni e si é Pa is-Saclay, E y, 91057, F ance
8Eu opean Molecula Biology Labo a o y, Eu opean Bioin o ma ics Ins i u e, Wellcome Genome Campus, Hinx on, Camb idge, CB10
1SD, UK
9SIB Swiss Ins i u e o Bioin o ma ics, Amphipôle, Qua ie UNIL-So ge, Lausanne, 1015, Swi ze land
Fi s published: 25 Sep 2025, 5:298
h ps://doi.o g/10.12688/open eseu ope.20937.1
La es published: 11 No 2025, 5:298
h ps://doi.o g/10.12688/open eseu ope.20937.2
2
Abs ac
Hanak's ba (Pipis ellus hanaki Hul a and Benda 2004) is one o he
mos ange es ic ed mammals in Eu ope, since i occu s only in
Cy enaica, Libya, and C e e (G eece). I is cu en ly classi ied as
'Vulne able' on he IUCN Red Lis , wi h i s o aging habi a h ea ened
by a numbe o human ac i i ies. The e e ence genome o Hanak's
ba (Pipis ellus hanaki) will p o ide a c ucial esou ce o unco e ing
he species phylogene ic his o y and will help assess he deg ee o
gene ic isola ion among i s popula ions. A o al o 23 con iguous
ch omosomal pseudomolecules (sex ch omosomes included) we e
Open Pee Re iew
App o al S a us
1 2
e sion 2
( e ision)
11 No 2025
iew
e sion 1
25 Sep 2025 iew
Richa d O on, Uni e si y o Glasgow, 1.
Open Resea ch Eu ope
Page 1 o 11
Open Resea ch Eu ope 2025, 5:298 Las upda ed: 19 NOV 2025
Co esponding au ho : Chia a Bo oluzzi ([email p o ec ed])
Au ho oles: Geo gakakis P: In es iga ion, Resou ces, W i ing – O iginal D a P epa a ion, W i ing – Re iew & Edi ing; Ka akasi D:
In es iga ion, Me hodology, P ojec Adminis a ion, Resou ces, Supe ision, W i ing – Re iew & Edi ing; Lymbe akis P: In es iga ion,
Resou ces, W i ing – Re iew & Edi ing; Papadimi akis M: In es iga ion, Resou ces, W i ing – Re iew & Edi ing; S a akis M:
In es iga ion, Resou ces, W i ing – Re iew & Edi ing; Bi zilekis E: In es iga ion, Resou ces, W i ing – Re iew & Edi ing; Poulakakis N:
In es iga ion, Resou ces, W i ing – Re iew & Edi ing; Böhne A: Me hodology, P ojec Adminis a ion, Supe ision, W i ing – Re iew &
Edi ing; Mon ei o R: Me hodology, P ojec Adminis a ion, Supe ision, W i ing – Re iew & Edi ing; Fe nández R: Me hodology, P ojec
Adminis a ion, Supe ision, W i ing – Re iew & Edi ing; Escude o N: Me hodology, P ojec Adminis a ion, Supe ision, W i ing – Re iew
& Edi ing; Moussy A: In es iga ion, Supe ision, W i ing – Re iew & Edi ing; C uaud C: In es iga ion, Supe ision, W i ing – Re iew &
Edi ing; Labadie K: In es iga ion, Supe ision, W i ing – O iginal D a P epa a ion; Demi djian L: Da a Cu a ion, Fo mal Analysis,
W i ing – Re iew & Edi ing; Mangeno S: Da a Cu a ion, Fo mal Analysis, W i ing – Re iew & Edi ing; Belse C: Da a Cu a ion, Fo mal
Analysis, W i ing – Re iew & Edi ing; Wincke P: In es iga ion, Supe ision, W i ing – Re iew & Edi ing; Oli ei a PH: In es iga ion,
Supe ision, W i ing – Re iew & Edi ing; Au y JM: Da a Cu a ion, Fo mal Analysis, Supe ision, W i ing – Re iew & Edi ing; Hagge y L:
Da a Cu a ion, Fo mal Analysis, W i ing – Re iew & Edi ing; Sinha S: Da a Cu a ion, Fo mal Analysis, W i ing – Re iew & Edi ing; Ma in F:
Da a Cu a ion, Fo mal Analysis, W i ing – Re iew & Edi ing; Bo oluzzi C: Visualiza ion, W i ing – O iginal D a P epa a ion
Compe ing in e es s: No compe ing in e es s we e disclosed.
G an in o ma ion: Biodi e si y Genomics Eu ope (G an no.101059492) is unded by Ho izon Eu ope unde he Biodi e si y, Ci cula
Economy and En i onmen call (REA.B.3); co- unded by he Swiss S a e Sec e a ia o Educa ion, Resea ch and Inno a ion (SERI) unde
con ac numbe s 22.00173 and 24.00054; and by he UK Resea ch and Inno a ion (UKRI) unde he Depa men o Business, Ene gy
and Indus ial S a egy’s Ho izon Eu ope Gua an ee Scheme. This wo k was suppo ed by he Genoscope, he Commissa ia à l'Éne gie
A omique e aux Éne gies Al e na i es (CEA), F ance Génomique (ANR-10-INBS-09-08), and he explo a o y esea ch p og amme
‘ATLASea: A las o ma ine genomes and i s a ge ed p ojec SEQ-Sea (ANR-22-EXAT-0003-SEQ-Sea).
The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Copy igh : © 2025 Geo gakakis P e al. 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, 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 ci ed.
How o ci e his a icle: Geo gakakis P, Ka akasi D, Lymbe akis P e al. ERGA-BGE e e ence genome o Hanak's ba ( Pipis ellus
hanaki), an IUCN Vulne able species es ic ed o o es -like bio opes [ e sion 2; pee e iew: 2 app o ed] Open Resea ch Eu ope
2025, 5:298 h ps://doi.o g/10.12688/open eseu ope.20937.2
Fi s published: 25 Sep 2025, 5:298 h ps://doi.o g/10.12688/open eseu ope.20937.1
assembled om he genome sequence. This ch omosome-le el
assembly encompasses 1.9 Gb, composed o 447 con igs and 141
sca olds, wi h con ig and sca old N50 alues o 48.7 Mb and 89.1 Mb,
espec i ely.
Keywo ds
Pipis ellus hanaki, genome assembly, Eu opean Re e ence Genome
A las, Biodi e si y Genomics Eu ope, Ea h Biogenome P ojec ,
Vespe ilionidae amily, Hanak's ba , Νανονυχτερίδα του Hanak
This a icle is included in he Ho izon Eu ope
ga eway.
This a icle is included in he Genome Repo s
om he Biodi e si y Genomics Eu ope P ojec
collec ion.
Glasgow, UK
Jiang Feng, No heas No mal Uni e si y,
Changchun, China
2.
Any epo s and esponses o commen s on he
a icle can be ound a he end o he a icle.
Open Resea ch Eu ope
Page 2 o 11
Open Resea ch Eu ope 2025, 5:298 Las upda ed: 19 NOV 2025
In oduc ion
Hanak’s ba (Pipis ellus hanaki Hul a and Benda 2004) o
Νανονυχτερίδα του Hanak in G eek, is one o he six wes -
Palaea c ic membe s o he genus Pipis ellus Kaup, 1829 s.s
(Figu e 1). Phylogene ically, i is he sis e species o Pipis ellus
pygmaeus (Leach, 1825). Hanak’s ba is a small ba , wi h a
pale b own o us y b own pelage colo a ion, mode a ely pale
and dis inc ly us ie han in he o he wo congene ic species,
Pipis ellus pipis ellus and Pipis ellus pygmaeus. The
ace, wing memb anes, ea s, and agi a e da k b own, whe eas
he ea bases and he a ea a ound he eyes a e sligh ly pale .
In Eu ope, only a small-sized o m o Hanak’s ba occu s in
C e e, P. hanaki c e icus Benda, 2009, while he nomino ypical
o m, P. hanaki hanaki Hul a and Benda, 2004 li es jus
in no he n Cy enaica, Libya. These wo o ms di e in
mo phome ic and gene ic ai s (Benda e al., 2004; Benda e al.,
2008; Benda e al., 2014); while P. hanaki hanaki is he la ges
membe o he common pipis elle (Pipis ellus pipis ellus)
g oup, P. hanaki c e icus has a signi ican smalle body and
skull size and i s endemic C e an popula ion was e en
sugges ed o ep esen a sepa a e species, since i s phylogene ic
posi ion was no ully esol ed, bu i was shown o be a sis e
species o bo h P. hanaki s.s and P. pygmaeus (Benda e al.,
2014). This phylogene ic econs uc ion, howe e , has no ye
been accep ed.
The Hanak’s ba was i s disco e ed in he no h-eas -
e n pa o Cy enaica, Libya (Benda e al., 2004) and was
subsequen ly ound solely in he cen e o he o es ed no he n
pa o he Cy enaican pla eau (Jebel Al Akhda M s). I s
p esence on C e e was i s documen ed in 2007 (Hul a e al.,
2007) om a specimen collec ed in he wes e n pa o he
island which was analysed mo phologically and gene ically.
Since hen, i has been ound in se e al locali ies, mos ly in
o es ed a eas, ee cul i a ions, and ege a ed we lands o
wes e n and cen al C e e (Benda e al., 2008; Geo giakakis
e al., 2023). Al hough a he widesp ead on C e e, Pipis ellus
hanaki was no loca ed in se e al sampling e o s ha we e
ecen ly unde aken in many islands o sou h-eas G eece, iz.
Rhodes and Ka pa hos (Geo giakakis e al., 2023).
Pipis ellus hanaki is cu en ly classi ied as ‘Vulne able’ on
he IUCN Red Lis (Geo giakakis e al., 2020), e lec ing i s
high isk o ex inc ion in he wild because o , among o he s,
signi ican habi a loss due o esiden ial and comme cial
de elopmen , o es i es and expansion o cul i a ions
(Geo giakakis e al., 2018). Addi ionally, i is lis ed unde
Annex IV o he Habi a s Di ec i e (92/43/EU) and Appendix
II o he Be n Con en ion highligh ing i s p o ec ion and
conse a ion s a us as a species o Eu opean in e es and
necessi a ing he p ope managemen o i s habi a .
Pipis ellus hanaki u ilizes a g ea a ie y o oos s ( ees,
ock issu es, buildings), bu i depends on ma u e ees –
na i e Que cus bu also cul i a ed Olea, Ce a onia, Ficus and
P unus – o o aging (Geo giakakis e al., 2018). As an insec
p eda o , i plays an impo an ole in he o es ecosys em
unc ion and esilience.
De eloping a high-quali y e e ence genome o Pipis ellus
hanaki is essen ial o ad ance ou unde s anding o i s unique
gene ic makeup. This genomic esou ce will also suppo
conse a ion e o s by p o iding aluable insigh s in o i s
phylogene ic ela ionships wi hin he P. pipis ellus g oup and
he gene ic di e ences be ween P. hanaki hanaki and P. hanaki
c e icus.
The gene a ion o his e e ence esou ce was coo dina ed by
he Eu opean Re e ence Genome A las (ERGA) ini ia i e’s Bio-
di e si y Genomics Eu ope (BGE) p ojec , suppo ing ERGA’s
aims o p omo ing ansna ional coope a ion o p omo e
ad ances in he applica ion o genomics echnologies o p o ec
and es o e biodi e si y (Mazzoni e al., 2023).
Ma e ials & me hods
ERGA’s sequencing s a egy includes Ox o d Nanopo e
Technology (ONT) and/o Paci ic Biosciences (PacBio)
o long- ead sequencing, along wi h Hi-C sequencing o
ch omosomal a chi ec u e, Illumina Pai ed-End (PE) o
polishing (i.e. ecommended o ONT-only assemblies), and
RNA sequencing o ansc ip ome p o iling, o acili a e
genome assembly and anno a ion.
Amendmen s om Ve sion 1
We hank he e iewe o hei commen s. We ha e add essed all
commen s in his new manusc ip submission. As sugges ed by
he e iewe , we also added a pic u e o a Hanak’s ba indi idual,
which is howe e di e en om he indi idual ha was used in his
Repo o he e e ence genome p oduc ion. This is speci ied in
he Figu e cap ion in he main ex o a oid misunde s anding.
Any u he esponses om he e iewe s can be ound a
he end o he a icle
REVISED
Figu e 1. A specimen o he species Pipis ellus hanaki. The
indi idual in he pic u e is a di e en specimen om he one
sampled and sequenced o his s udy. Pho o c edi : Panagio is
GEORGIAKAKIS, Na u al His o y Museum o C e e, C e e.
Page 3 o 11
Open Resea ch Eu ope 2025, 5:298 Las upda ed: 19 NOV 2025
Sample and sampling in o ma ion
On 21 Oc obe 2023, one adul male o Pipis ellus hanaki was
sampled by Panagio is Geo gakakis om he Na u al His o y
Museum o C e e (NHMC) o he Uni e si y o C e e. The
species was iden i ied using he iden i ica ion key o Die z &
Kie e (2016). The specimen was collec ed wi h a mis nes in
Psilo i is m ., Rou as o es , I akleio, C e e, G eece. Sampling
was pe o med unde P esiden ial Dec ee 67/1981 issued by
he G eek Go e nmen . The specimen was eu hanized by
inc easing concen a ion o CO2, a e which i was immedia ely
lash- ozen in liquid ni ogen, and p ese ed a -80°C un il
DNA ex ac ion.
Vouche ing in o ma ion
Physical e e ence ma e ial o he he e sequenced specimen
has been deposi ed in he Ve eb a es Collec ions o he
NHMC h ps://www.nhmc.uoc.g /en/depa men s/ e eb a es
unde accession ID NHMC.80.5.121.52.
F ozen e e ence issue ma e ial o muscle and li e is
a ailable om he same indi idual a he Genomics and Gene ic
Resou ces Di ision o he NHMC h ps://www.nhmc.uoc.g /en/
depa men s/genomics unde accession ID NHMC.80.5.121.52.
Gene ic in o ma ion
The es ima ed genome size, es ima ed by Genomes on a T ee
(GoaT) (Challis e al., 2023) by ances al s a e econs uc ion,
is 2.12 Gb. This is a diploid genome wi h a haploid numbe o
22 ch omosomes (2n=44). All in o ma ion o his species
was e ie ed om GoaT.
DNA/RNA p ocessing
DNA was ex ac ed om muscle (45 mg) using a Genomic- ip
100/G Ki (QIAGEN, MD, USA) ollowing manu ac u e
ins uc ions. DNA agmen size selec ion was pe o med using
Sho Read Elimina o (PacBio, CA, USA). Quan i ica ion
was pe o med using a Qubi dsDNA HS Assay ki (The mo
Fishe Scien i ic) and in eg i y was assessed in a Fem oPulse
sys em (Agilen ). DNA was s o ed a 4 °C un il usage.
RNA was ex ac ed om 10 mg o muscle using he RNeasy
Plus Uni e sal ki (Qiagen) ollowing manu ac u e ins uc ions.
Residual genomic DNA was emo ed wi h 6U o TURBO
DNase (2 U/μL) (The mo Fishe Scien i ic). Quan i ica ion was
pe o med using a Qubi RNA HS Assay ki and in eg i y was
assessed in a Bioanalyze sys em (Agilen ). RNA was s o ed
a -80 °C.
Lib a y p epa a ion and sequencing
Long- ead DNA lib a ies we e p epa ed wi h he SMRTbell
p ep ki 3.0 ollowing manu ac u e s’ ins uc ions and
sequenced on a Re io sys em (PacBio). Hi-C lib a ies we e
gene a ed om muscle (20 mg) o he same indi idual using
he A ima High Co e age HiC ki ( ollowing he Animal
Tissues low inpu p o ocol 01) and sequenced on a No aSeq
6000 ins umen (Illumina) wi h 2x150 bp ead leng h. Poly(A)
RNA-Seq lib a ies we e cons uc ed using he Illumina S anded
mRNA P ep, Liga ion P ep ki (Illumina) and sequenced
on an Illumina No aSeq X Plus ins umen (Illumina)
wi h 2x150 bp ead leng h. In o al, 36x PacBio and 19x HiC
da a we e sequenced o gene a e he assembly.
Genome assembly me hods
The genome o Pipis ellus hanaki was assembled using he
Genoscope GALOP pipeline (h ps://wo k lowhub.eu/wo k-
lows/1200). B ie ly, aw PacBio HiFi eads we e assembled
using Hi iasm 0.19.5- 593 (Cheng e al., 2021). Remaining
allelic duplica ions we e emo ed using pu ge_dups 1.2.5
(Guan e al., 2020) wi h de aul pa ame e s and he p oposed
cu o s. The pu ged assembly was sca olded using YaHS
1.2 (Zhou e al., 2023) and assembled sca olds we e hen
cu a ed h ough manual inspec ion using P e ex View 0.2.5 o
emo e alse joins and inco po a e sequences no au oma i-
cally sca olded in o hei espec i e loca ions wi hin he ch o-
mosomal pseudomolecules. Summa y analysis o he eleased
assembly was pe o med using he ERGA-BGE Genome
Repo ASM Galaxy wo k low (10.48546/wo k lowhub.wo k-
low.1104.1).
Genome anno a ion me hods
A gene se was gene a ed using he Ensembl Gene Anno a ion
sys em (Aken e al., 2016), p ima ily by aligning sho - ead
RNA-seq da a om BioSample SAMEA115120717 o a
p e ious e sion o he e e ence genome (GCA_964339955.1).
Gaps in he anno a ion we e illed ia p o ein- o-genome
alignmen s o a selec se o e eb a e p o eins
om UniP o (UniP o Conso ium, 2019), which had
expe imen al e idence a he p o ein o ansc ip le el. A
each locus, da a we e agg ega ed and consolida ed, p io i ising
models de i ed om RNA-seq da a, esul ing in a inal se
o gene models and associa ed non- edundan ansc ip
se s. To dis inguish ue iso o ms om agmen s, he likelihood
o each open eading ame (ORF) was e alua ed agains
known e eb a e p o eins. Low-quali y ansc ip models, such
as hose showing e idence o agmen ed ORFs, we e emo ed.
In cases whe e RNA-seq da a we e agmen ed o absen ,
homology da a we e p io i ised, a ou ing longe ansc ip s wi h
s ong in on suppo om sho - ead da a. The esul ing gene
models we e classi ied in o h ee ca ego ies: p o ein-coding,
pseudogene, and long non-coding. Models wi h hi s o known
p o eins and ew s uc u al abno mali ies we e classi ied as
p o ein-coding. Models wi h hi s o known p o eins bu
displaying abno mali ies, such as he absence o a s a
codon, non-canonical splicing, unusually small in on s uc u es
(<75 bp), o excessi e epea co e age, we e eclassi ied
as pseudogenes. Single-exon models wi h a co esponding
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Open Resea ch Eu ope 2025, 5:298 Las upda ed: 19 NOV 2025
Figu e 2. Snail plo summa y o assembly s a is ics. The main plo is di ided in o 1,000 size-o de ed bins a ound he ci cum e ence,
wi h each bin ep esen ing 0.1% o he 1,892,181,625 bp assembly. The dis ibu ion o sequence leng hs is shown in da k g ey, wi h he
plo adius scaled o he longes sequence p esen in he assembly (211 Mb, shown in ed). O ange and pale-o ange a cs show he sca old
N50 and N90 sequence leng hs (89,115,682 and 49,289,172 bp), espec i ely. The pale g ey spi al shows he cumula i e sequence coun
on a log-scale, wi h whi e scale lines showing successi e o de s o magni ude. The blue and pale-blue a ea a ound he ou side o he plo
shows he dis ibu ion o GC, AT, and N pe cen ages in he same bins as he inne plo . A summa y o comple e, agmen ed, duplica ed, and
missing BUSCO genes ound in he assembled genome om he Mammalia da abase (odb10) is shown on he op igh .
mul i-exon copy elsewhe e in he genome we e classi ied as
p ocessed ( e o ansposed) pseudogenes. Models ha did no
i any o he p e iously desc ibed ca ego ies did no o e lap
p o ein-coding genes and we e cons uc ed om ansc ip omic
da a we e conside ed po en ial lncRNAs. Po en ial lncRNAs
we e u he il e ed o emo e single-exon loci due o hei
un eliabili y. Pu a i e miRNAs we e p edic ed by pe o ming
a BLAST sea ch o miRBase (Kozoma a e al., 2019)
agains he genome, ollowed by RNA old analysis (G ube
e al., 2008). O he small non-coding loci we e iden i ied by
scanning he genome wi h R am (Kal a i e al., 2018) and
passing he esul s h ough In e nal (Naw ocki & Eddy, 2013).
Resul s
Genome assembly
The genome assembly has a o al leng h o 1,892,181,625 bp
in 141 sca olds (Figu e 2 and Figu e 3), wi h a GC con en o
42.5%. I ea u es a con ig N50 o 48,700,701 bp (L50=16)
and a sca old N50 o 89,115,682 bp (L50=7). The e a e 306
gaps, o alling 34.1 kb in cumula i e size. The single-copy gene
con en analysis using he Mammalia da abase wi h BUSCO
(Manni e al., 2021) esul ed in 93.3% comple eness (92.0%
single and 1.3% duplica ed). 96.3% o eads k-me s we e
p esen in he assembly and he assembly has a base accu acy
Quali y Value (QV) o 61.4 as calcula ed by Me qu y (Rhie e al.,
2020).
Genome anno a ion
The genome anno a ion consis s o 18,099 p o ein-coding
genes wi h associa ed 30,313 ansc ip s, in addi ion o 5,633
non-coding genes (Table 1). Using he longes iso o m pe
ansc ip , he single-copy gene con en analysis using he
Mammalia odb10 da abase wi h BUSCO esul ed in
95.3% comple eness. Using he OMAme Me azoa- 2.0.0.h5
da abase o OMA k (Ne e s e al., 2025) esul ed in 95.7%
comple eness and 98.5% consis ency (Table 2).
Page 5 o 11
Open Resea ch Eu ope 2025, 5:298 Las upda ed: 19 NOV 2025
Table 1. S a is ics om assembled gene models.
No.
genes
No.
ansc ip s
Mean gene
leng h (bp)
No. single-
exon genes
Mean
exons pe
ansc ip
mRNA 18,099 30,313 41,209 1,346 11.4
pseudogene 386 386 14,593 33 13.3
snoRNA 972 972 123 972 1.0
lncRNA 3,043 3,204 3,256 2,630 1.4
miRNA 135 135 82 135 1.0
snRNA 1,263 1,263 119 1,263 1.0
RNA 110 110 225 110 1.0
scRNA 77 77 148 77 1.0
O he ncRNA 33 33 96-281 33 1.0
Table 2. Anno a ion comple eness and consis ency sco es calcula ed by BUSCO un
in p o ein mode (mammalia_odb10) and OMA k (Me azoa- 2.0.0.h5).
Comple e Single copy Duplica ed F agmen ed Missing
BUSCO 8,790 (95.3%) 8,706 (94.4%) 84 (0.9%) 74 (0.8%) 362 (3.9%)
OMA k 13,499 (95.7%) 13,207 (93.7%) 292 (2.0%) - 596 (4.2%)
Consis en Inconsis en Con aminan s Unknown
OMA k 17,942 (98.5%) 197 (1.1%) 0.0 (0.0%) 81 (0.4%)
Figu e 3. Hi-C con ac map showing spa ial in e ac ions be ween egions o he genome. The diagonal co esponds o
in a-ch omosomal con ac s, depic ing ch omosome bounda ies. The equency o con ac s is shown on a loga i hmic hea map scale.
Hi-C ma ix bins we e me ged in o a 50 kb bin size o plo ing. Due o space cons ain s on he axes, only he GenBank names o he
18 h la ges au osomes and he Y ch omosome (GenBank: OZ203255.1) a e shown.
Page 6 o 11
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Benda P, Geo giakakis P, Die z C, e al.: Ba s (Mammalia: Chi op e a) o he
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2896–2898.
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Kal a i I, Naw ocki EP, A gasinska J, e al.: Non-coding RNA analysis using he
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sequences o unc ion. Nucleic Acids Res. 2019; 47(D1): D155–D162.
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MazzoniCJ,Cio iC,Wa e houseRM:Biodi e si y: an a las o Eu opean
Da a a ailabili y
Pipis ellus hanaki and he ela ed genomic s udy we e assigned
o T ee o Li e ID (ToLID) ‘mPipHan1’ and all sample, sequence,
and assembly in o ma ion a e a ailable unde he umb ella
BioP ojec PRJEB77247. The sample in o ma ion is a ailable
a he ollowing BioSample accessions: SAMEA115799862,
SAMEA115799867, and SAMEA115120717. The genome
assembly is accessible om ENA unde accession numbe
GCA_964339955.4 and he anno a ed genome is a ailable a
he Ensembl websi e (h ps://p ojec s.ensembl.o g/e ga-bge/).
Sequencing da a p oduced as pa o his p ojec a e a ail-
able om ENA a he ollowing accessions: ERX12737184,
ERX12737185, ERX14169058, ERX14169059, and
ERX12733454. Documen a ion ela ed o he genome assembly
and cu a ion can be ound in he ERGA Assembly Repo
(EAR) documen a ailable a h ps://gi hub.com/ERGA-conso -
ium/EARs/ ee/main/Assembly_Repo s/Pipis ellus_hanaki/
mPipHan1. Fu he de ails and da a abou he p ojec a e
hos ed on he ERGA po al a h ps://po al.e ga-biodi e si y.eu/
da a_po al/412090.
Au ho con ibu ions
DK coo dina ed he p ojec ; PG collec ed he species; PG
iden i ied he species; DK, MP, MS, EB, PL, and NP sampled
and p ese ed biological ma e ial and p o ided me ada a;
AsB, RM, RF, and NE p o ided suppo in sampling, shipping
o biological ma e ial, me ada a collec ion, and managemen ;
GST ex ac ed DNA, p epa ed lib a ies, and pe o med sequenc-
ing unde he supe ision o AM, CC, KL, PHO, and PW;
SM, CB, LD, and JMA pe o med genome assembly and cu a-
ion unde he supe ision o JMA; LH, SS, and FM pe o med
genome anno a ion; CB gene a ed he analysis and epo .
All au ho s con ibu ed o he w i ing, e iew, and edi ing o
his genome no e and ead and app o ed he inal e sion.
This wo k is pa o he species assigned o Genoscope, which
was ins umen al in he we lab, sequencing, and assembly
p ocesses, and ep esen s a key con ibu ion o BGE’s ou pu s.
Au ho in o ma ion
Membe s o he Genoscope Sequencing Team a e lis ed
he e: h ps://zenodo.o g/ eco ds/14611490.
Acknowledgemen s
We would like o exp ess ou g a i ude o M. Kan za idou
and I. Ekklisia chos o assis ance in ieldwo k. We acknowl-
edge he suppo o he F eibu g Galaxy Team: Saim Momin
and Bjö n G üning, Bioin o ma ics, Uni e si y o F eibu g
(Ge many), unded by he Ge man Fede al Minis y o Educa ion
and Resea ch BMBF g an 031 A538A de.NBI-RBC and he
Minis y o Science, Resea ch and he A s Baden-Wü embe g
(MWK) wi hin he amewo k o LIBIS/de.NBI F eibu g. We
would like o acknowledge he assembly e iewe , Tyle S.
Alio o om he Cen o Nacional de Análisis Genómico
(CNAG).
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e e ence genomes. Na u e. 2023; 619(7969): 252.
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Bioin o ma ics. 2013; 29(22): 2933–2935.
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Ne e s Y, Wa wick Vesz ocy A, Rossie V, e al.: Quali y assessmen o gene
epe oi e anno a ions wi h OMA k. Na Bio echnol. 2025; 43(1): 124–133.
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Rhie A, Walenz BP, Ko en S, e al.: Me qu y: e e ence- ee quali y,
comple eness, and phasing assessmen o genome assemblies. Genome
Biol. 2020; 21(1): 245.
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UniP o Conso ium: UniP o : a wo ldwide hub o p o ein knowledge. Nucleic
Acids Res. 2019; 47(D1): D506–D515.
PubMed Abs ac | Publishe Full Tex | F ee Full Tex
Zhou C, McCa hy SA, Du bin R: YaHS: Ye ano he Hi-C Sca olding ool.
Bioin o ma ics. 2023; 39(1): b ac808.
PubMed Abs ac | Publishe Full Tex | F ee Full Tex
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Open Pee Re iew
Cu en Pee Re iew S a us:
Ve sion 2
Re iewe Repo 19 No embe 2025
h ps://doi.o g/10.21956/open eseu ope.23670. 63933
© 2025 Feng J. This is an open access pee e iew epo dis ibu ed unde he e ms o he C ea i e Commons
A ibu ion License, 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 ci ed.
Jiang Feng
No heas No mal Uni e si y, Changchun, China
This manusc ip p esen s a high-quali y ch omosome-le el e e ence genome assembly and
anno a ion o Pipis ellus hanaki—a IUCN Vulne able species wi h a na ow dis ibu ion. The s udy
ollows s anda dized genomic wo k lows, including PacBio HiFi sequencing and Hi-C sca olding,
and p o ides comp ehensi e assembly s a is ics and anno a ion de ails, which is aluable o
en ich genomic esou ce o his species and suppo ing i s conse a ion. The e a e wo issues
ega ding species axonomic cla i y and me hodological a ional need o be add essed o enhance
he manusc ip ’s scien i ic igo and p ac ical u ili y.
1.A c i ical inconsis ency exis s be ween he manusc ip ’s i le and he ac ual sequenced
subspecies. The i le e e s o Hanak's ba (Pipis ellus hanaki), bu he In oduc ion explici ly s a es
ha he sequenced indi idual is om C e e, belonging o he subspecies P. hanaki c e icus (no he
nomino ypical P. hanaki hanaki om Libya). Mo eo e , he manusc ip ails o add ess whe he he
genome o P. hanaki c e icus can ully ep esen he en i e species, especially gi en p e ious
deba es abou he C e an popula ion po en ially being a dis inc species (Benda e al., 2014). This
ambigui y unde mines he gene alizabili y o he " e e ence genome" and con uses eade s abou
i s axonomic scope. The e o e, he au ho s a e sugges ed o e ise he i le o explici ly e lec he
subspecies, e.g., "ERGA-BGE e e ence genome o he C e an subspecies o Hanak's ba (Pipis ellus
hanaki c e icus), an IUCN Vulne able species es ic ed o o es -like bio opes". And add a
dedica ed subsec ion in he Discussion o add ess: 1) gene ic di e ences be ween P. hanaki
c e icus and P. hanaki hanaki; 2) limi a ions o using a single subspecies o ep esen he en i e
species, and ecommenda ions o u u e genome sequencing o he Libyan subspecies o enable
in e -subspecies compa isons.
2. The Me hods sec ion s a es ha gene anno a ion elied on "publicly a ailable sho - ead RNA-
seq da a om BioSample SAMEA115120717" aligned o a p e ious e sion o he genome
(GCA_964339955.1), a he han RNA-seq da a gene a ed om he newly assembled genome in
his s udy. No explana ion is p o ided o his choice, why public da a was p io i ized. This aises
conce ns abou anno a ion accu acy, as public da a may no pe ec ly ma ch he new assembly o
he speci ic C e an subspecies. I own RNA-seq da a was gene a ed bu unused, explain echnical
Open Resea ch Eu ope
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