This document summarizes analysis of structural variant detection in individual NA24385 using electronic DNA mapping. It describes: 1) The Nabsys electronic mapping workflow and strategies for variant verification by mapping to reference vs discovery through de novo assembly. 2) Analysis of 2783 putative deletions in NA24385, with 491 evaluated and 151 confirmed at specificity ≥0.9. 3) Examples of confirmed deletions, insertions, and outliers, showing concordance between measured and expected sizes. 4) The ability of de novo assembly to accurately reconstruct small genomes and detect structural variants.

1. Analysis of NA24385 v0.5.0

2. Electronic Mapping
2
Sequencing-by-Synthesis (SBS)
DNA Sample
Electronic Mapping
DNA Sample
DNA Fragmentation Labeling Chemistry
50-300+ kb<800 bp

3. Nabsys Workflow
3
High molecular weight
DNA isolation
35-500 kb molecules
Sequence-specific tag
attachment
Tagged sample introduction
into instrument
Single-molecule tag detection at
a velocity of >1 Mbp/s.
t
V
DNA
backbone
Sequence-specific tags

4. SV Detection Strategies – Verification vs. Discovery
4
Variant Verification
Mapping to
Reference
SV Discovery
De Novo Map
Assembly

5. Hypothesis-Based Deletion Verification
5
fusion point
fusion point
breakpoints
breakpoint
breakpoint
GRCh37 ref
GRCh37 ref
ALT reference segment
ALT reference segment
Probe pattern change
Interval size change

6. Application of SV-Verify to Evaluate Putative NA24385 Deletions
6
Kaiser, M.D. et al. bioRxiv doi: 10.1101/140699
Oliver, J.S. et al. bioRxiv doi: 10.1101/139840
14 – 40 terms
SV-Verify
300 – 499 bp
500 – 999 bp
≥1000 bp
Multi-interval
ROC
curves
HD-Mapping
Reference
(GRCh37)
Putative
deletions
Specificity of
Detection
Single-molecule
reads

7. Analysis of Deletions in v0.5.0
• 2783 Deletions ≥300 bp were evaluated
• One deletion is considered in cases where multiple deletions are called
in the same interval.
• Breakpoints must have a minimum number of “reads” that span the
area and have sufficient information content.
• 491 evaluated deletions are not postulated in HG002. 151 have
specificity scores ≥0.9.
• Single technology calls:
• PacBio: 363
• ILMN: 106
• 10X: 4
• CG: 0
7

8. 0
200
400
600
800
1000
1200
1400
300-499 bp
500-999 bp
1000-3000 bp
>3000 bp
#Deletions
Size Range
Deletion Size Distribution
Not confirmed
Confirmed sp=0.9
Evaluated Deletion Size Distribution
8

9. 58%
30%
70%
12%
0
50
100
150
200
250
300
PB 300-1000 bp
ILMN 300-1000 bp
PB >1000 bp
ILMN >1000 bp
#Deletions
Technology and size
Verification of PacBio Only or ILMN Only Calls
Not Confirmed
Confirmed sp=0.9
Single Technology Calls
9

10. Deletions Supported in HG002
10
Of the 491 evaluated deletions that are postulated in HG003/
HG004 but not called in HG002, 151 deletions (31%) are
called by us in HG002 and have a specificity score ≥ 0.90.
0
20
40
60
80
100
120
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
#Deletions
Specificity score
Specificity Distribution

11. Evidence for 345 bp Deletion in HG002
Confidential 11
HG4_PB_assemblyticsfalcon_10975

12. Concordance of Deletions
12
y = 1.001x
R² = 0.99987
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
MeasuredDeletionSize
Expected Deletion Size
Measured Deletion Size vs. Expected Size

13. Concordance of Deletions
13
y = 1.001x
R² = 0.99987
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
MeasuredDeletionSize
Expected Deletion Size
Measured Deletion Size vs. Expected Size

14. Outliers
14
y = 1.001x
R² = 0.99987
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
MeasuredDeletionSize
Expected Deletion Size
Measured Deletion Size vs. Expected Size
-4788 bp
-1941 bp
+2847 bp
1922 bp

15. Outliers
15
y = 1.001x
R² = 0.99987
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
MeasuredDeletionSize
Expected Deletion Size
Measured Deletion Size vs. Expected Size
-998 bp
-2547 bp
-2047 bp
-100 bp
1068 bp

16. Accurate De novo Assembled Genome Maps of Small Genomes
16
Alignment of Nabsys Bordetella pertussis assembly with NCBI reference map
y = 0.996x + 0.3097
R² = 0.9995
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
AssemblyConsensusIntervalSize(bp)
NCBI Reference Interval Size (bp)
300
500
700
900
1100
1300
1500
1700
1900
300
800
1300
1800
Average interval spacing = 3.2 kb

17. SV Detection Strategies – Verification vs. Discovery
17
Variant Verification
Mapping to
Reference
SV Discovery
De Novo Map
Assembly

18. De Novo 1 kb Insertion
18
98.24 98.26 98.28 98.30 98.32 98.34 98.36
map2796
GRCh37 chr 9
Measured insertion size: 1039 bp
Predicted insertion sizes: 1023, 1006 bp
x 106!
98.24 98.26 98.28 98.30 98.32 98.34 98.36
map2796
HG2_PB_SVrefine2PBcRplusDovetail_1500

19. De Novo 686 bp Deletion and 583 bp Insertion
19
map879
134.30 134.32 134.34 134.36 134.38 134.40
GRCh37 chr 10
x 106
Measured insertion size: 583 bp
Predicted SV sizes: 441, 37, -37, 38,
37, 220 bp
Measured deletion size: -686 bp
Predicted SV sizes: -51, -527, -28,
-153, -627, -34, -793 bp
HG3_Ill_MetaSV_694

20. De Novo 12607 bp Insertion of Four Probes
20
66.36 66.38 66.40 66.42 66.44 66.46 66.48 66.50 66.52 66.54
map118
GRCh37 chr 15
x 106
Measured insertion size: 12,607 bp
Predicted SV size: 12,136 bp ( )
HG2_PB_PB10Xdip_10768

21. Verification vs. Discovery
21
“What we observe is not nature herself, but nature exposed to our
method of questioning.”
- Werner Heisenberg
Variant Verification
Mapping to
Reference
SV Discovery
De Novo Map
Assembly