Supplementary Figures
Figure S1: Simulation flowchart. Step 1: First, 2 founder labels are assigned to each pedigree
founder for our selection of 318,609 markers. In this example of Family A, a total of 8
founder labels are thus assigned for the 8 first markers of a chromosome. Then, Mendelian
transmission and the recombination process are simulated according to the genetic distance
between markers. In this example, we observed two cross-overs, one between label 3 and
label 4, one between label 5 and label 6. Step 2: One of the 738 European 1,000 genomes
haplotypes (1000G) is randomly drawn without replacement for this chromosome and
assigned to a founder label. Here, 4 haplotypes of 8 loci have been drawn for founder labels
1, 5, 6 and 8. The alleles in red come from a SNP chip (Affymetrix 250K chip), and the ones in
blue are frequent polymorphisms present in WES data. Step 3: SNP chip and WES genotype
data are created. Markers with homozygous genotypes for the reference alleles for all the
individuals are removed from WES genotype data (alleles in grey here). Step 4: Linkage
analyses are performed with reference, SNP chip and WES genotype data. Before performing
linkage analysis with genotype data, SNPs are removed to minimize the LD. Then, for each
linkage analysis, the genome was divided into 3 regions: linked, non-informative (in grey)
and
excluded
(Ex.).
80,938 SNVs
9,301 INDELs
Missense, nonsense, splice-site, and INDEL
frameshifts
14,498 SNVs
2,572 INDELs
Genotypes coherent with the disease model
(All individuals heterozygous or not covered)
1,420 SNVs
227 INDELs
Frequency below 0.1 %
in 3 reference samples
6 SNVs
3 INDELs
Figure S2: WES filtering strategy and data for Family A. The reference samples are the
European of 1,000 genomes project, the European American of exome variant server, and a
control database of IntegraGen.
42,964 SNVs
3,336 INDELs
Missense, nonsense, splice-site, and
INDEL frameshifts
13,215 SNVs
2,459 INDELs
All individuals heterozygous
or not covered
All individuals homozygous
or not covered
3,144 SNVs
535 INDELs
2,423 SNVs
408 INDELs
Frequency below 1 %
in 3 reference samples
Frequency below 1 %
in 3 reference samples
6 SNVs
1 INDEL
101 SNVs
2 INDELs
Gene with at least 2
candidate variants
2 SNVs
0 INDEL
Figure S3: WES filtering strategy and data for Family B. The reference samples are the
European of 1,000 genomes project, the European American of exome variant server, and a
control database of IntegraGen.
(For legend see next page.)
Figure S4: Marker selection in simulated datasets. Each boxplot shows values computed on
100 replicates, with a marker selection according to different physical length bins. Red and
blue lines represent the median of values computed with all the markers (i.e. without
marker selection). The second row (Non-Informative) shows the genetic length of the
genome with a LOD score computed on genetic data between -2 and the linkage threshold
(0.8, 2.6 and 1.1 for Families A, B and B-nuclear, respectively). The third row (False Positive)
shows the genetic length of the genome with a LOD score computed on the genetic data
higher than the linkage threshold, and with a REF LOD score below -2. The fourth row (False
Negative) shows the genetic length of the genome with a LOD score computed on the
genetic data below -2, and with a REF LOD higher than the linkage threshold. The fifth row
(True Positive) shows the genetic length of the genome with a REF LOD score and a LOD
score computed on the genetic data higher than the linkage threshold. The last row (True
Negative) shows the genetic length of the genome with a REF LOD score and a LOD score
computed on the genetic data below -2.
Figure S5: Strong linkage disequilibrium leads to false negative signals in Family A. Figure
(A) shows an example of haplotype inheritance in Family A. Each color represents a different
founder origin. Here, we observe a linkage region, surrounded in green, with a purple
haplotype shared by all affected individuals. Figure (B) shows haplotype reconstruction by a
multipoint approach, such as Merlin, in presence of linkage disequilibrium (LD). In situation
of strong LD, haplotype frequencies are incorrectly estimated because they are estimated by
multiplying allele frequencies together: it is thus more likely to observe a single rare
haplotype (the one in green in individual A3), than to observe 3 rare haplotypes with 2
recombinations.
Figure S6: Exclusion by linkage analysis of candidate heterozygous variants B8 and B9,
located on the same haplotype, in the recessive Family B. This figure shows the haplotype
reconstruction obtained with Merlin around the heterozygous candidate variants of Family B
from both SNP chip and WES genotype data. Each color represents a different founder
origin. Here, only one haplotype is shared identical by descent (the orange one), proving that
the
2
candidate
variants
are
on
this
haplotype.
Supplementary Tables
Table S1: Characteristics of SNP chip and WES markers used in the simulations
Intermarker distances (bp)
# markers
All markers
WES
SNP chip
Family A
WES
a
a
SNP chip
Family B
a
SNP chip
a
WES a
1st decile
Median
9th decile
# gaps
> 250 kb > 500 kb > 1000 kb
248,290
166
4,879
27,402
137
48
26
71,206
47
2,952
67,524
2,169
974
384
132,804
254
8,631
49,119
538
106
30
43,981
67
5,212
121,022
2,358
1,109
455
248,290
166
4,879
27,402
137
48
26
43,225
70
5,440
124,949
2,358
1,106
454
the average of numbers of markers, distances and gaps used in the 100 replicates are presented.
Table S2: Linkage analysis performance in simulated datasets before minimizing linkage
disequilibrium
SNP chip
WES
Exclusion
NI
Linkage
Exclusion
NI
Linkage
b
Exclusion 2834.75
312.56
10.51
2316.91
840.08
7.46
NI
0.85
1.33
0.53
0.72
8.98
0.48
c
Linkage
14.39
138.88
283.2
7.49
208.7
220.55
2849.99 458.77 294.24
2325.12 1057.76 228.49
Exclusion 3156.68 233.67
1.32
3133.75
249.98
0.04
NI
10.39
207.95
2.14
0.79
218.55
0.10
d
Linkage
0.00
0.23
7.22
0.00
2.15
5.26
3167.06 441.85
10.68
3134.54
470.68
5.40
Exclusion 3065.61 289.43
27.73
2992.75
376.13
6.06
NI
0.01
6.11
0.81
0.08
6.26
0.36
e
Linkage
0.02
3.16
226.72
0.25
27.53
201.20
3065.64 298.71 255.26
2993.07
409.92
207.62
a
reference LOD score; b genetic length in cM; c linkage threshold = 0.8; d linkage threshold = 2.6;
linkage threshold = 1.1.
Family
B-nuclear
Family B
Family A
REF a
e
Table S3: Characteristics of the WES candidate variants of Families A and B
Family A
Family B e
Family
B-nuclear e
Type
Genotypes a
LOD score
SNP chip data b
LOD score
WES data b
Sanger
sequencing c
Variant A1
SNV
HET/HET/HET
[0.90 ; 0.90]
[0.85 ; 0.85]
Validated
Variant A2
SNV
HET/HET/HET
[0.90 ; 0.90]
[0.90 ; 0.90]
Validated
Variant A3
SNV
HET/HET/HET
[0.90 ; 0.90]
[0.90 ; 0.90]
Validated
Variant A4
SNV
HET/HET/HET
[0.86 ; 0.86]
[0.88 ; 0.88]
Validated
Variant A5
SNV
HET/HET/HET
[0.89 ; 0.89]
[0.90 ; 0.90]
Validated
Variant A6
SNV
HET/NC/NC
[-2.51 ; -2.93]
[-3.38 ; -3.38]
Not validated
Variant A7
INDEL
HET/HET/HET
[-2.59 ; -2.70]
[-1.43 ; -1.46]
Not validated
Variant A8
INDEL
HET/HET/HET
[-4.38 ; -4.41]
[-4.11 ; -4.02]
Inconclusive d
Variant A9
INDEL
HET/HET/HET
[-3.07 ; -2.94]
[-3.10 ; -3.08]
Not validated
Variant B1
SNV
HOM/NC/NC
[-2.39 ; -3.23]
[-2.52 ; -2.05]
Not validated
Variant B2
SNV
HOM/NC/NC
[-5.83 ; -5.59]
[-2.49 ; -2.49]
Not validated
Variant B3
SNV
HOM/NC/NC
[-3.16 ; -5.68]
[-INF ; -INF]
Not validated
Variant B4
SNV
HOM/NC/NC
[-5.34 ; -5.31]
[-INF ; -INF]
Not validated
Variant B5
SNV
HOM/HOM/HOM
[2.61 ; 2.61]
[2.61 ; 2.61]
Validated
Variant B6
SNV
HOM/NC/NC
[-10.78 ; -INF]
[-INF ; -INF]
Not validated
Variant B7
INDEL
HOM/HOM/HOM
[2.61 ; 2.61]
[2.59 ; 2.61]
Inconclusive
Variant B1
SNV
HOM/NC/NC
[-1.80 ; -2.57]
[-1.99 ; -1.37]
Not validated
Variant B2
SNV
HOM/NC/NC
[-4.70 ; -4.46]
[-1.69 ; -1.69]
Not validated
Variant B3
SNV
HOM/NC/NC
[-2.55 ; -5.68]
[-INF ; -INF]
Not validated
Variant B4
SNV
HOM/NC/NC
[-4.76 ; -4.74]
[-INF ; -INF]
Not validated
Variant B5
SNV
HOM/HOM/HOM
[1.20 ; 1.20]
[1.20 ; 1.20]
Validated
Variant B6
SNV
HOM/NC/NC
[-10.12 ; -INF]
[-INF ; -INF]
Not validated
INDEL
HOM/HOM/HOM
[1.20 ; 1.20]
[1.18 ; 1.20]
Inconclusive
SNV
HET/HET/HET
[-INF ; -5.79]
[-INF ; -INF]
Validated
Variant B7
Variant B8
f
f
Variant B9
SNV
HET/HET/HET
[-5.79 ; -5.78]
[-INF ; -INF]
Validated
b
genotypes of the candidate variants for the 3 affected individuals of each Family; LOD scores of
the 2 closest markers bracketing the candidate variants; c validation status by Sanger sequencing; d
inconclusive INDELs are located in stretches of Poly-A/Poly-T, which sequences are difficult to
interpret; e variants B1 to B7 of Family B and Family B-nuclear are the same; f variants located on the
same gene.
HOM: homozygote; HET: heterozygote; NC: not covered; INF: infinity.
a