ARTICLE
Au oMap is a high pe o mance homozygosi y
mapping ool using nex -gene a ion sequencing
da a
Ma hieu Quinodoz1,2,3, Vi ginie G. Pe e 1,2,3,4, Nicola Bedoni5, Bé yl Roye Be and5, Ka a ina Cisa o a5,
A ash Salmaninejad 6, Neda Sepahi7, Raquel Rod igues8, Meh an Pi an7,9, Majid Moja ad6,
Ali eza Pasda 6,10, Ali Ghanba i Asad 7, Ana Be a Sousa8,11, Luisa Cou inho San os 12,
And ea Supe i-Fu ga 5& Ca lo Ri ol a 1,2,3✉
Homozygosi y mapping is a powe ul me hod o iden i ying mu a ions in pa ien s wi h
ecessi e condi ions, especially in consanguineous amilies o isola ed popula ions. His o i-
cally, i has been used in conjunc ion wi h geno ypes om highly polymo phic ma ke s, such
as DNA mic osa elli es o common SNPs. T adi ional so wa e pe o ms a he poo ly wi h
da a om Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS), which
a e now ex ensi ely used in medical gene ics. We de elop Au oMap, a ool ha is bo h web-
based o downloadable, o allow pe o ming homozygosi y mapping di ec ly on VCF (Va ian
Call Fo ma ) calls om WES o WGS p ojec s. Following a aining s ep on WES da a om 26
consanguineous amilies and a alida ion p ocedu e on a ma ched coho , ou me hod shows
highe o e all pe o mances when compa ed wi h eigh exis ing ools. Mos impo an ly,
when es ed on eal cases wi h nega i e molecula diagnosis om an in e nal se , Au oMap
de ec s h ee gene-disease and mul iple a ian -disease associa ions ha we e p e iously
un ecognized, p ojec ing clea benefi s o bo h molecula diagnosis and esea ch ac i i ies in
medical gene ics.
h ps://doi.o g/10.1038/s41467-020-20584-4 OPEN
1Ins i u e o Molecula and Clinical Oph halmology Basel (IOB), Basel, Swi ze land. 2Depa men o Oph halmology, Uni e si y o Basel, Basel, Swi ze land.
3Depa men o Gene ics and Genome Biology, Uni e si y o Leices e , Leices e , UK. 4Ins i u e o Expe imen al Pa hology, Lausanne Uni e si y Hospi al
(CHUV), Lausanne, Swi ze land. 5Se ice o Medical Gene ics, Lausanne Uni e si y Hospi al (CHUV), Lausanne, Swi ze land. 6Depa men o Medical
Gene ics, Facul y o Medicine, Mashhad Uni e si y o Medical Sciences, Mashhad, I an. 7Noncommunicable Diseases Resea ch Cen e , Fasa Uni e si y o
Sciences, Fasa, I an. 8Depa men o Medical Gene ics, Hospi al San a Ma ia, Cen o Hospi ala Uni e si á io Lisboa No e (CHULN), Lisbon Academic
Medical Cen e (CAML), Lisbon, Po ugal. 9Bioin o ma ics and Compu a ional Biology Resea ch Cen e , Shi az Uni e si y o Medical Sciences, Shi az, I an.
10 Di ision o Applied Medicine, Medical School, Uni e si y o Abe deen, Abe deen, UK. 11 Medical Facul y, Lisbon Uni e si y, Lisbon, Po ugal. 12 Ins i u o de
O almologia D Gama Pin o, Lisbon, Po ugal. ✉email: [email p o ec ed]
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1234567890():,;
Homozygosi y mapping (HM), also called au ozygosi y
mapping, is a echnique aimed a de ec ing and sco ing
he p esence o consecu i e homozygous geno ypes, o
“ uns o homozygosi y”(ROHs) in a pe son’s genome. ROHs
esul om he co-inhe i ance o po ions o DNA ha a e p e-
alen in a gi en popula ion, and no mally ange om a ew o
hund eds o megabases (Mb), depending on he e hnic g oup
conside ed1,2. In he o sp ing o consanguineous unions, ROHs
co espond in la ge pa o he gene ic ma e ial ha is co-
inhe i ed om ances o s who a e common o bo h pa en s3. Fo
ins ance, child en o pa en s who a e fi s cousins ha e ROHs o
DNA egions ha we e in he e ozygosis in hei g ea -g and-
pa en s, and we e inhe i ed om bo h hei ma e nal and
pa e nal sides.
In pa ien s wi h ecessi e condi ions, especially i belonging o
a consanguineous pedig ee, ROHs e y o en encompass he
mu a ion ha is esponsible o he disease4, also o igina ing om
a heal hy he e ozygous common ances o , and he e o e a e
excellen p oxy ma ke s o he mu a ion i sel . In addi ion, HM
can also be used o de ec la ge he e ozygous dele ions esul ing
in hemizygous (and appa en ly homozygous) geno ypes, as well
as o cases o unipa en al disomy5,6. Fo hese easons, HM has
been used o decades in medical gene ics as a ool o iden i y
egions o he genome o be p io i ized o a ge ed mu a ional
sc eens. In he pas , HM p ocedu es elied on geno ypes om
polymo phic ma ke s ( ypically mic osa elli es) o de e mine
whe he pa ien s had consecu i e homozygous calls s. heal hy
ela i es. Since he beginning o his cen u y, he comme ciali-
za ion o high-densi y mic oa ays o single-nucleo ide poly-
mo phisms (SNPs) has allowed he use o he SNP geno ypes o
he same pu pose, by in e oga ing usually non-coding a ian s
wi h an ele a ed deg ee o he e ozygosi y in he gene al popula-
ion. Se e al so wa e we e hen de eloped, such as PLINK7,
Homozygosi yMappe 8, o GERMLINE9. In i ue o he e y
high accu acy o hei calls (>99.8%)10, SNP mic oa ays ou pu s
a e highly eliable o iden i y ROHs. Howe e , hey do no p o-
ide any in o ma ion on a pa ien ’s mu a ions, since hese la e
DNA a ian s a e usually a e and he e o e a e no included in
such a ays.
Con e sely, in o ma ion om whole-exome sequencing
(WES), which is la gely used in con empo a y medical gene ics,
allows he disco e y o any ype o DNA a ian s, including
equen geno ypes and a e mu a ions alike. The e o e, a leas in
p inciple, WES can be used as a single echnology o bo h HM
and mu a ion de ec ion. Howe e , HM algo i hms de eloped o
a ay echnologies end o deli e sub-op imal esul s when
applied o WES, since hey a e no adap ed o handle he in insic
noise (2.53–30.60%)10 ha is ypical o his sequencing
me hod11–13. In pa icula , ROHs de ec ed om WES da a wi h
his so wa e a e smalle in size and p oduce lowe cumula i e
au ozygosi y alues, o en esul ing in he end in alsely-nega i e
ou pu 14–16. Hence, many in es iga o s s ill make use o wo
sepa a e ools: SNP mic oa ays o de e mine ROHs, and WES o
iden i y possible a e mu a ions, wi h an impo an was e o ime
and esou ces17–21.
To ci cum en his p oblem, in his wo k we p esen Au oMap
(Au ozygosi y Mappe ), a so wa e ha can p o ide e y eliable
HM esul s di ec ly om s anda d WES ou pu s and is applicable
o WGS (whole-genome sequencing) as an addi ional ea u e, and
compa e i wi h o he ools, including some ha we e specifically
de eloped o pe o m HM on WES da a (BCFTools16, FILTUS22,
H3M223, HOMWES15, Sa yVc Homozygosi y, and Sa yHo-
mozygosi y24). Each o he ools used in his compa ison ha e
hei own s ongpoin s and limi a ions, such as he p esence o a
g aphical use in e ace (GUI), a c oss-pla o m ope a i e sys em,
a use - iendly ou pu ype, e c25,26. Au oMap is accessible bo h
ia a web in e ace ( ee o cha ge), o allow low- h oughpu o
occasional use o exome da a, and as a command-line ool, o
la ge-scale genomic p ojec s, o WGS, and o allow in eg a ion
in o ou ine analy ical pipelines.
Resul s and discussion
Au oMap akes as an inpu a ian call o ma (VCF) files
(Fig. 1a), i.e., s anda d esul files om a a ie y o commonly-
used so wa e o analyzing WES and WGS sequences ( a ian
calle s), con aining he lis o DNA a ian s de ec ed in a gi en
sample wi h espec o he human e e ence genome. VCF files
ha e he ad an age o being o smalle size compa ed o p ima y
mapping files (BAM files), con ain mos o he in o ma ion
ha can be used o HM pu poses and, impo an ly, a e gene ally
mo e a ailable o he end-use s, such as biologis s o physicians.
O no e, single-sample VCFs a e accep ed by bo h web and
s andalone e sions o Au oMap, while mul isample VCFs a e
only accep ed by he s andalone e sion. Once he file is uploaded
ia he web in e ace (h ps://au omap.iob.ch/) he use simply
launches he analysis, which in he end p oduces an ou pu such
as he one indica ed in Fig. 1b, c. This includes a PDF file showing
he g aphical ep esen a ion o au ozygous egions along he
au osomes, as well as a ex file epo ing he same in o ma ion as
nume ical alues, wi h he posi ions o he de ec ed ROHs, hei
size, numbe o a ian s, and pe cen age o homozygosi y.
Au oMap can also include he X ch omosome, i eques ed by he
use , o bo h emales and males. In emales, his ch omosome is
ea ed as an au osome, whe eas in males all hemizygous calls a e
conside ed as homozygous. In addi ion, i is possible o p o ide a
lis o genes (o a “gene panel”, e.g., a lis o genes linked o a
gi en condi ion) as a ex file, o immedia ely ecognize whe he
hey a e o no wi hin a gi en ROH (Fig. 1c). The same p ocedu e
can also be pe o med on a local compu e , by di ec ly down-
loading he sou ce files (h ps://gi hub.com/mquinodo/
Au oMap). In i s s andalone e sion, Au oMap does no need
compiling bu equi es he ins alla ion o some addi ional so -
wa e (BCFTools, 1.9 o la e ; BEDTools, 2.25.0 o la e ; Pe l,
5.22.0 o la e ; R, 3.2.0 o la e ).
As men ioned, one o he key poin s o he p ope de ec-
ion o ROH om WES da a is he a ge ed emo al o alse
posi i e a ian calls. These calls no only ac as a noise wi h
espec o ue signals, bu also ac i ely lead o an a ificial
agmen a ion o ROHs (when a he e ozygous alse posi i e
call is p esen in a uly au ozygous egion) o e en o he
comple e miss o a ROH ( o egions wi h a low numbe o
calls). To his end, as a fi s s ep, all a ian s om he VCF file
a e ca e ully assessed wi h c i e ia elying on co e age and
al e na i e eads coun . Mo e p ecisely, hey a e e alua ed
acco ding o hei sequencing dep h, he a io be ween eads
aligned o he e e ence sequence s. al e na i e alleles ( o
he e ozygous calls), and o hei loca ion in epea ed egions
o he genome (Fig. 1a and Supplemen a y Table 1). Va ian s
ha do no sa is y hese s ingen c i e ia a e selec i ely and
indi idually elimina ed om u he analyses. Impo an ly, all
such c i e ia we e e ified no o be specific o any gi en
a ian calling so wa e, wi h he aim o enabling analyses o
VCF files p oduced by a ious p og ams.
A e his fil e ing s ep a he a ian le el, ROHs a e iden ified
by a sliding-window app oach, associa ed wi h ou unable
pa ame e s: he size o he window, he minimal numbe o
homozygous a ian s in he window, he maximal gap be ween
wo consecu i e a ian s in one ROH, and he maximal size o
ex ension om he bounda ies o s e ches o de ec ed ROHs
(Fig. 1a and Supplemen a y Table 1). Following hei iden ifica-
ion, ROHs a e hen selec ed only i hey each a minimal alue in
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e ms o size, numbe o a ian s and pe cen age o homozygous
geno ypes in he egion (Fig. 1a and Supplemen a y Table 1).
To e alua e he pe o mances o Au oMap, we ga he ed da a
om 52 amilies wi h ecessi e he edi a y blindness, o which
we also had geno ypes om bo h WES and SNP a ays, and
selec ed one pa ien pe amily. These indi iduals we e all om
consanguineous pedig ees li ing in Po ugal o in I an, i.e., in
coun ies wi h a low and an ele a ed deg ee o consanguini y a
he le el o he gene al popula ion, espec i ely27,28. Mo e p e-
cisely, hey all had a median cumula i e ROH size o 234.8 Mb
(de ec ed by PLINK on SNP a ay da a), anging be ween 90.6
and 819.5 Mb. We hen spli hese indi iduals in o wo ma ched
se s o 26 pe sons each, hus defining a aining coho and a
ma ched alida ion coho (Supplemen a y Da a 1). As a e e -
ence o ue posi i e alues, we adop ed he me hod desc ibed by
Kanche a e al.15, consis ing in he use o he PLINK so wa e7
applied wi h de aul pa ame e s on da a om SNP a ays.
We op imized he pa ame e s o Au oMap on he aining
coho (Supplemen a y Table 1 and Supplemen a y Fig. 1A, B, see
“Me hods”sec ion), and hen used he op imized alues o ana-
lyze he alida ion coho . We pe o med s abili y analyses by
a ying each pa ame e indi idually, which esul ed in small
a ia ion o he pe o mances, indica i e o he obus ness o he
me hod (Supplemen a y Fig. 1C). In addi ion, he a e age pe -
o mance was no significan ly di e en be ween he wo coho s,
demons a ing no o e -fi ing o he pa ame e s on he aining
da a (Supplemen a y Fig. 2A). Mo e p ecisely, specifici y in he
aining and alida ion se s had alues o 77.5% and 79.6%,
espec i ely (p=0.35, unpai ed - es ), while sensi i i y
had alues o 90.5% and 92.4%, espec i ely (p=0.18).
We also compa ed he e ec o h ee mains eam a ian
calle s ha could be adop ed by Au oMap end use s (GATK-
Haplo ype Calle , Sam ools-mpileup and S elka) on he pe o -
mance o ou ool (based on da a om he aining se ). Au o-
Map’s sensi i i y was sligh ly lowe when S elka was used
(−1.4% compa ed o Haplo ype Calle ), whe eas specifici y
appea ed o be sligh ly lowe when Sam ools was used, (−4.4%
compa ed o Haplo ype Calle , Supplemen a y Fig. 2B). These
di e ences we e all below 5%, he e o e indica ing ha ou ool is
o e all insensi i e o he choice ope a ed by a po en ial use wi h
espec o a gi en calling so wa e.
Nex , we compa ed Au oMap wi h eigh p e iously-published
ools, namely: PLINK applied on exome da a7, Homo-
zygosi yMappe o WES29, HOMWES15, BCFTools/RoH16,
FILTUS22,H
3M223, Sa yHomozygosi y, and Sa yVc Homo-
zygosi y24 on da a om he alida ion coho , composed o 26
indi iduals wi h a ious le els o au ozygosi y (Supplemen a y
Fig. 3). All o hem we e based on a hidden Ma ko model wi h
he excep ion o Homozygosi yMappe , PLINK, and HOMWES,
which we e de eloped by applying a sliding window me hod (as i
is he case o Au oMap as well). O no e, H3M2and Sa yHo-
mozygosi y canno p ocess VCF files bu need a BAM file
(sequence alignmen da a), Homozygosi yMappe is only a ail-
able ia a web in e ace, and FILTUS can only be que ied ia a
Fig. 1 Au oMap wo kflow and example o ou pu . a Wo kflow ollowed by Au oMap wi h de aul se ings, b,cexample o g aphical and ex ou pu o
pa ien NSI-326, wi h he ollowing pa ame e s: DP =8, pe cal low =0.25, pe cal high =0.75, binomial =0.000001, maxgap =10, window =7,
window h es =5, minsize =2, min a =25, minpe c =88, ch X =No, and ex end =1. Blue egions ep esen de ec ed ROHs.
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g aphical use in e ace (GUI), no allowing au oma ed p oces-
sing o mul iple files. The use Sa yHomozygosi y and Sa -
yVc Homozygosi y equi es he downloading o p e-p epa ed
linkage da a om 1000 Genomes p ojec o a join c om a ew
hund ed WGS. We did no include o he so wa e packages such
as Agile-Geno ype , Agile-Va ian Mappe 14, and HomSI30 since
hey do no p o ide a genome-wide esul file, which could be
used as sou ce o da a o compa ison, bu only a g aphical
ou pu . We used de aul pa ame e s o all hese ools (i.e., he
pa ame e s op imized by hei espec i e au ho s o analyze WES
da a), excep o PLINK, since his so wa e was fi s de eloped
o SNP a ay da a and he e o e had o be e-pa ame ized o
allow i s use on WES geno ypes (see “Me hods”sec ion).
Au oMap and Sa yVc Homozygosi y had he bes sensi i i y/
specifici y combina ion and he highes F-sco e when compa ed
o o he so wa e (p< 3.0 × 10−7, pai ed - es ) (Fig. 2a, b).
Howe e , Au oMap had a significan ly highe sensi i i y han
Sa yVc Homozygosi y (p< 2.9 × 10−6) and lowe specifici y
(p< 2.8 × 10−6).
In addi ion, Au oMap (on WES) was he ool ha displayed he
closes alues, in e ms o ROHs size and numbe , o he e e ence
(i.e., PLINK on SNP a ay) (Fig. 3). This indica ed no o e y low
ROH agmen a ion, especially in compa ison o o he ools,
which p oduced o ins ance ei he a high numbe o small and
agmen ed ROHs (H3M2, FILTUS, and PLINK) o de ec ed only
la ge ROHs (Sa yVc Homozygosi y). Two ep esen a i e
examples o his e ec , o e en i e cho omosomes, a e p esen ed
in Supplemen a y Fig. 4.
Since small ROHs a e mo e di ficul o de ec and indeed could
penalize he global pe o mance o some o hese ools, we
epea ed he same analysis by conside ing only ROHs la ge han
5 Mb, being awa e ha such egions a e mo e likely o ha bo
causa i e a ian s o ecessi e diseases4. As expec ed, e e y ool
displayed inc eased pe o mances, due o he a ificial clea ing o
Fig. 2 Pe o mance o Au oMap and o he ools on da a om he alida ion se . a,bPe o mance o Au oMap o ROHs la ge han 1 Mb (Megabase):
aspecifici y and sensi i i y; bF-sco e. c,dSame analyses as in a,b, bu limi ed o ROHs wi h sizes o 5 Mb o highe (mos likely o con ain causa i e
ecessi e mu a ions in medical gene ics p ac ice, acco ding o published li e a u e). E o ba s ep esen s anda d de ia ions o he mean. Fo boxplo s
(b,d), he middle band indica es he median, boxes ep esen he fi s and he hi d qua iles, and whiske s indica e he la ges obse a ion smalle han o
equal o he fi s qua ile −1.5 x IQR ( he in e qua ile ange) and he smalles obse a ion g ea e han o equal o o hi d qua ile +1.5 x IQR. N=
~2.7 million DNA a ian s pe ool and pe es . Sou ce da a a e p o ided in he Sou ce Da a File.
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smalle ROHs, and Au oMap, Sa yHomozygosi y and Sa -
yVc Homozygosi y had he bes F-sco es (p< 2.0 × 10−4, Fig. 2c,
d). Au oMap had a significan highe sensi i i y han Sa yVc -
Homozygosi y (p=2.9 × 10−6) bu lowe han Sa yHomo-
zygosi y (p< 6.9 × 10−9). Conce ning specifici y, he esul s we e
e e sed, wi h Au oMap ha ing a significan ly highe alue han
Sa yHomozygosi y (p< 3.0 × 10−3) bu a lowe sco e wi h
espec o Sa yVc Homozygosi y (p< 2.5 × 10−5). In summa y,
Au oMap had he bes pe o mances o e all when es ed on ou
alida ion coho . Since his coho comp ised only indi iduals o
Eu opean and Sou h Asian ances y, u he assessmen s may be
needed o e alua e popula ion-specific pe o mances on da a
om o he e hnic g oups. In addi ion, a possible con ounding
ac o in ou analysis could be ep esen ed by he use o di e en
cap u e ki s o WES. Al hough we did no assess his pa ame e
a he expe imen al le el, p e ious li e a u e has shown ha
conco dance a es o called a ian s ac oss di e en cap u e ki s
a e e y ele a ed31, making subs an ial a iabili y o pe o mance
linked o he use o di e en ki s a a he unlikely e en . Con-
ce ning WGS da a, addi ional es s will be also needed o
benchma k exis ing ools such as PLINK o o he a ay-specific
so wa e s. Au oMap, due o he di e ence in e ms o numbe
o a ian s and co e age compa ed o WES.
In addi ion o an inc eased pe o mance in e ie ing ROHs
om exome da a, Au oMap also displayed o he benefi s. Fo
ins ance, Au oMap was he only ool ha could be un bo h ia a
web applica ion ( o he a e age use ) and he command-line, o
allow mo e compu a ionally-inclined use s o exploi i s ull
po en ial. In addi ion, he a e age p ocessing ime displayed by
Au oMap was ela i ely sho , especially in ela ionship o i s
pe o mance. All ools, excep hose using BAM files as an inpu
(H3M2and Sa yHomozygosi y) could analyze a s anda d VCF
in 30 s o less. The as es ools we e BCFTools and PLINK,
comple ing he analysis in less han 5 s, while Au oMap ook
app oxima ely 20 s. A mo e de ailed compa ison o all hese
ea u es is p o ided in Supplemen a y Table 2.
As a final es , we used Au oMap on unexplo ed WGS and
WES esea ch da a om ou labo a o y, o cases wi h unknown
molecula e iology. Following expe imen al alida ion, ou ool
was ins umen al in he end o iden i ying bo h coding and
noncoding mu a ions, all included in o la ge ROHs, o a ew
new synd omes. These included: an in e genic dele ion causing
de elopmen al de ec s32, a small dele ion a ec ing splicing in he
gene PISD and esul ing in he Libe a b synd ome33, and a
pa ial duplica ion o he NMNAT1 gene, esponsible o a new
mul isys em diso de 34. Compa ed o he o he ools analyzed,
Au oMap was he only one allowing he comple e de ec ion o he
au ozygous egions con aining he mu a ions in all h ee cases
(Supplemen a y Table 3). Fo he NMNAT1 s udy, o example,
he lack o pe o mance displayed by some o he ools was likely
due o he p esence o a ew alse-posi i e he e ozygous a ian s
in he ROH con aining he pa hogenic duplica ion, which we e
indeed ecognized as such—and hence disca ded—by Au oMap.
The same was ue o he wo o he s udies, whe e he p esence
o alse-posi i e he e ozygous geno ypes was combined wi h he
ela i ely sho size o ROHs and/o he p esence o a limi ed
numbe o iden ifiable geno ypes. In addi ion, Au oMap enabled
he de ec ion o new a ian s in a numbe o genes ha we e
al eady associa ed wi h Mendelian condi ions35–37. In conclusion,
Au oMap is a eliable ool ha can p edic ROHs wi h high
specifici y and sensi i i y, in less han a minu e, e en om VCF
files de i ed om noisy exome sequencing da a. I is a ailable
bo h ia a web-based in e ace, o a quick analysis, as well as a
command-line package, allowing la ge-scale and ou ine analyses.
Me hods
Pa ien s and DNA. This s udy adhe ed o he o he ene s o he Decla a ion o
Helsinki and was app o ed by he Ins i u ional Re iew Boa ds o ou espec i e
ins i u ions: he E hikkommission No dewes - and Zen alschweiz (2019-01660),
he Ins i u ional Re iew Boa ds o Mashhad Uni e si y o Medical Sciences
(961015), he e hics commission o he Oph halmic Hospi al “D Gama Pin o”in
Lisbon (16.05.20) and he Noncommunicable Diseases Resea ch Cen e o Fasa
Uni e si y o medical sciences (IR.FUMS.REC.1396.211). W i en in o med con-
sen o ms we e signed by all subjec s, ec ui ed a he Oph halmic Hospi al “D
Gama Pin o”in Lisbon, and a he Fasa and Mashhad Uni e si ies o Medical
Science in I an. Pa icipan s had ei he epo ed consanguini y o had ~100 Mb o
mo e o cumula i e ROHs as de e mined by PLINK applied on a ay da a (Sup-
plemen a y Da a 1). Genomic DNA was ex ac ed om pe iphe al blood leuko-
cy es. Mo e p ecisely, o Po uguese pa ien s DNA ex ac ion was pe o med by
using he EZ1 DNA blood ki and EZ1 DNA bu y coa ca d (Qiagen), acco ding o
he manu ac u e ’s ins uc ions, choosing an elu ion olume o 200 μl. Fo I anian
pa ien s, DNA was ex ac ed om blood using RPN8512 Nucleon BACC3 DNA
Ex ac ion Ki (Illus a).
A ay geno yping. DNA o s udied indi iduals we e geno yped a he iGE3
Pla o m o he Uni e si y o Gene a, Swi ze land, using Illumina Infinium a ays
(San Diego, USA; GSAMD-24 2.0, GSA-24 2.0, Co eExome-24 1.1, and
Co eExome-24 1.2). Geno ypes alues we e ob ained wi h GenomeS udio
(Illumina).
Exome sequencing. Exome cap u e and lib a y p epa a ion was pe o med using
he Su eSelec Human All Exon 6 ki (Agilen , San aCla a, USA) and HiSeq Rapid
PE Clus e Ki 2 (Illumina, San Diego, USA) wi h 2 μg genomic DNA. Lib a ies
we e sequenced on a HiSeq 2500 o a No aSeq 6000 ins umen s (Illumina)
(Supplemen a y Da a 1). Raw eads we e mapped o he human genome e e ence
sequence (build hg19) wi h he No oalign so wa e (V3.08.00, No oc a Tech-
nologies, Selango , Malaysia). Duplica e eads we e hen emo ed using Pica d
( . 2.14.0-SNAPSHOT). Base quali y sco e ecalib a ion was pe o med and a ian
calling was done wi h Haplo ypeCalle (GATK, .4.0.3.0).
Au oMap equi emen s. Au oMap is composed o Bash, Pe l and R sc ip s. I
equi es BCFTools (≥ 1.9), BEDTools (≥ 2.25.0), Pe l (≥ 5.22.0), and R (≥ 3.2.0).
The ollowing e sions we e used o all analysis: BCFTools ( 1.9-78-gb7e4ba9),
BEDTools ( 2.25.0), Pe l ( 5.22.0), Bash (4.3.48(1)- elease) and R ( 3.5.1).
To be p ocessed by Au oMap, a VCF file mus con ain he GT (geno ype) and
AD (allelic dep hs o he e and al alleles) o DP4 (numbe o high-quali y e -
o wa d bases, e - e e se, al - o wa d, and al - e e se bases) fields.
F
H
V
S
A
3
P
WB
0
4
8
12
0 40 80 120 160 200
A e age numbe o ROHs
Median o ROH size a e ages [Mb]
Au oMap
BCFTools
Fil us
H3M2
HOMWES
A
B
F
3
W
Homozygosi yMappe
PLINK
Sa yHomozygosi y
Sa yVc Homozygosi y
SNP−a ay PLINK
H
P
S
V
*
Fig. 3 A e age numbe o ROH de ec ed pe sample (N=26) s. he
median o he size a e ages o ROHs pe sample (ROHs la ge han
1Mb).The as e isk indica es he e e ence alue used o compa ison, i.e.,
da a om SNP a ays analyzed wi h PLINK. E o ba s ep esen s anda d
de ia ions o he mean. Sou ce da a a e p o ided in he Sou ce Da a File.
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Au oMap algo i hm. The fi s s ep o he algo i hm emo es a ian s om he
VCF file ha a e loca ed in epea egions, as epo ed by he UCSC genome
b owse (h ps://genome.ucsc.edu/cgi-bin/hgTables). Va ian s a e hen fil e ed by
quali y, based on he pe cen age o al e na i e eads (de aul op ions: –minpe cal
0.25 and –maxpe cal 0.75), on a binomial es o al e na i e and e e ence ead
coun s o he e ozygous a ian s (de aul op ion: –binomial 0.000001), and on
dep h (de aul op ion: –DP 8). A his poin , he analysis is s opped i he e a e less
han 10,000 a ian s su i ing such p ocedu es.
ROHs a e subsequen ly de ec ed by a sliding window wi h a fixed size and a
h eshold based on he numbe o homozygous a ian s (de aul op ions: –window
7 a ian s and –window h es 5). A e ha , de ec ed ROHs a e immed a hei
ends o emo e he e ozygous a ian s and a e ex ended a bo h ends (de aul
op ion: –ex end 1 [Mb]). ROHs con aining egions wi hou a ian s o a s e ch
la ge han a gi en h eshold (de aul op ion: –maxgap 10 [Mb]) a e spli in o wo
di e en ROHs by excluding he egion wi h no a ian s. Finally, ROHs a e fil e ed
o ha e a minimal size, a minimal numbe o a ian s and a minimal pe cen age o
homozygous a ian s (de aul op ions: –minsize 1 [Mb], –min a 25, and –minpe c
88 [%]).
P obabili ies o binomial dis ibu ion. P obabili ies o binomial dis ibu ions
we e calcula ed wi h he Pe l sc ip w i en by T.J. Finney (h ps://www.halo ype.
com/RKM/figu es/TJF/binomial. x ).
O e lap o ROHs. O e lap o ROHs we e ob ained wi h bed ools and he ol-
lowing command:
bed ools in e sec -a a.bed -b b.bed
Pa ame e s used o de ec ROHs om a ay da a. PLINK was used wi h de aul
pa ame e s, excep o a ian s wi h mino allele coun o 2 pe ba ch, o fil e ing
agains e y a e a ian s, o en ep esen ing alse-posi i e38,39:
plink –bfile bfile –homozyg –ou ou –homozyg-window-he 1 –homozyg-densi y
50 –homozyg-gap 1000 –homozyg-window-missing 5 –homozyg-window-snp 50 –
homozyg-snp 100 –homozyg-window- h eshold 0.05 –homozyg-kb 1000 –mac 2
De ec ion o ROHs om exome da a. All a ailable ools we e used wi h de aul
pa ame e s o ea ing inpu files, op imized by hei de elope s, excep o
PLINK. Fo his ool, we ook he pa ame e s defined by Kanche a e al. 15, o
op imize sensi i i y, and e- es ed he mos impo an pa ame e , i.e., he numbe
o allowed he e ozygous SNP pe ROH. This pa ame e is impo an o ake in
accoun he noise in exome sequencing da a. Indeed, low alues o his pa ame e
esul ed in low sensi i i y. We chose o use a alue o 3 since i p oduced a highe
sensi i i y wi h accep able specifici y (Supplemen a y Fig. 5).
The command used was:
plink –bfile bfile –homozyg –ou ou –homozyg-kb 1000 –homozyg-window-he 3
–homozyg-densi y 10000 –homozyg-gap 10000 –homozyg-window-missing 10 –
homozyg-window-snp 20 –homozyg-snp 10 –homozyg-window- h eshold 0.05
Fo Sa yHomozygosi y and Sa yVc Homozygosi y, we used he p e-p epa ed
linkage da a om 1000 Genomes p ojec , p o ided by he de elope s (h ps://
gi hub.com/ demolgen/Sa ySui e).
Pa ame e used o a ian calle s. Sam ools:
sam ools mpileup - DP,AD -ug e . as a inpu .bam|bc ools call - mO -o ou .
c
S elka:
configu eS elkaGe mlineWo kflow.py –bam inpu .bam – e e enceFas a e . as a
–exome
Haplo ypeCalle :
ga k –ja a-op ions “-Xmx4g”Haplo ypeCalle -R e . as a –dbsnp dbsnp. c -I
$inpu .bam -O ou . c
A e calling, a ian s we e fil e ed o be p esen in he egions included in he
cap u e ki s, wi h a 100 bp padding on each side.
Compu a ions o pe o mances. As a e e ence o ue posi i e alues, we
adop ed he me hod desc ibed by Kanche a e al.15, consis ing in he use o he
PLINK so wa e7applied wi h de aul pa ame e s on da a om SNP a ays. Sen-
si i i y and specifici y we e hen compu ed as ollows:
Sensi i i y %ðÞ¼ROHexomeall o e lapping wi h ROHa ay il e ed=To al ROHa ay il e ed;
Speci ici y %ðÞ¼ROHexome il e ed o e lapping wi h ROHa ayall=To al ROHexome il e ed;
whe e fil e ed ROHs a e la ge han 1 o 5 Mb and excluding gap egions such as
cen ome es, elome es, sho a ms, and he e och oma in defined om he gap
able in UCSC Table B owse (h ps://genome.ucsc.edu/cgi-bin/hgTables).
F-sco e was also added as a measu e o accu acy, compu ed as:
Fsco e ¼2sensi i i y speci ici y=sensi i i y þspeci ici yðÞ:
S a is ical es s.T- es we e pe o med wi h . es unc ion in RS udio ( 1.0.153)
wi h R ( 3.5.1).
Fo pai ed es : . es (x, y, al e na i e =“ wo.sided”, pai ed =TRUE)
Fo unpai ed es : . es (x, y, al e na i e =“ wo.sided”, pai ed =FALSE)
Figu es. Figu es we e done wi h ggplo 2 ( 3.3.2) and g idEx a ( 2.3) packages in
RS udio ( 1.0.153) wi h R ( 3.5.1).
Repo ing summa y. Fu he in o ma ion on esea ch design is a ailable in he Na u e
Resea ch Repo ing Summa y linked o his a icle.
Da a a ailabili y
A ay geno yping da a and exome sequencing da a canno be sha ed because o
es ic ions ela ed o he p o ec ion o pe sonal da a, as pe Swiss and Eu opean law.
Da a om Figs. 2and 3can be e ie ed in he Sou ce Da a file. Sou ce da a a e p o ided
wi h his pape .
Code a ailabili y
Au oMap code is eely a ailable on Gi Hub (h ps://gi hub.com/mquinodo/Au oMap/)
and was deposi ed in he zenodo.o g eposi o y (h ps://doi.o g/10.5281/
zenodo.4279125)40.
Recei ed: 17 Ma ch 2020; Accep ed: 9 Decembe 2020;
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Acknowledgemen s
This wo k was suppo ed by he Swiss Na ional Science Founda ion (g an # 176097,
o C.R.) and by he PhD Fellowships in Li e Science o he Uni e si y o Lausanne
( o M.Q.).
Au ho con ibu ions
M.Q. and C.R. designed he esea ch, w o e he manusc ip , analyzed all he da a,
and in e p e ed he esul s. V.P., N.B., A.S., N.S., R.R., M.P., M.M., A.P., A.G.A., A.B.S.,
and L.C-.S. pa icipa ed o he collec ion o biological samples and DNA ex ac ion.
V.P., N.B., B.R.B., K.C., and A.S.-F. pa icipa ed o he expe imen al design and p o ided
commen s.
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Addi ional in o ma ion
Supplemen a y in o ma ion is a ailable o his pape a h ps://doi.o g/10.1038/s41467-
020-20584-4.
Co espondence and eques s o ma e ials should be add essed o C.R.
Pee e iew in o ma ion Na u e Communica ions hanks Ryan Dhindsa, Sla é Pe o ski
and he o he , anonymous, e iewe o hei con ibu ion o he pee e iew o
his wo k.
Rep in s and pe mission in o ma ion is a ailable a h p://www.na u e.com/ ep in s
Publishe ’s no e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a filia ions.
Open Access This a icle is licensed unde a C ea i e Commons
A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing,
adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e
app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e
Commons license, and indica e i changes we e made. The images o o he hi d pa y
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