This report introduced main players, technical details, clinical service models in the field of NIPT both worldwide and within China.

1. Market & Tech analysis report
Non-Invasive Prenatal Testing
Produced by Li Ma, Msc, PhD candidate 2015

2. NIPT (Non-Invasive Prenatal Testing)
什么是无创产前检测 (NIPT) ?
通过采集孕妇的外周血，提取游离DNA (cell free DNA)，从而
安全准确的检测常见的胎儿非整倍性染色体异常。
- 唐氏综合征（T21）
- 爱德华氏综合征（T18）
- 帕陶氏综合征（T13）

3. Incidence of common chromosome abnormalities
发病原因 疾病名称 发病率
21号染色体三体
唐氏综合症
Down Sydrome 1 ：800
18号染色体三体
爱德华氏综合症
Edward Syndrome 1 : 3500 – 8000
13号染色体三体
帕陶氏综合症
Patau Syndrome
1 : 25 000

4. Facts about Down Syndrome
年龄 发病率
< 35 < 0.3 %
37 0.5 %
40 1 %
50 10 %
- 最常见的染色体非整倍体疾病(1:800)
- 先天性智力低下，生长发育迟缓，并伴发多系统畸形
- 随年龄增加发病率增加，>35岁直线上升

5. Screening and diagnosis of DS at clinical setting
94%-96% 5%-6%

6. Screening and diagnosis of DS at clinical setting

7. 无创产前筛查 (NIPT)
1997 : Lo et al. : cff DNA in maternal circulation
10-20 % of cell-free
DNA in maternal
circulation is fetal in
origin

8. 无创产前筛查 (NIPT)

9. Shotgun Massively Parallel Sequencing (s-MPS)
Gregg, A. R., et al. (2014). Annu Rev Genomics Hum Genet

10. Importance of fetal fraction
Fetal Fraction
Expected ratio
for Trisomy
4% 1.02
10% 1.05
20% 1.10
40% 1.20
Shotgun Massively Parallel Sequencing (s-MPS)

11. Shotgun Massively Parallel Sequencing (s-MPS)
Z-score

12. Distribution of %ChrNreference
%ChrNreference
Mean %ChrNreference
euploid T21
3σ
Z-score < 3 Z-score > 3
Shotgun Massively Parallel Sequencing (s-MPS)
Z-score

13.  Case-control study including 1696 pregnancies
 Detection rate (Sensitivity) – 98.6% (209/212)
 False-positive rate: 0.2% (3/1471)
 0.8% risk of test failure
Shotgun Massively Parallel Sequencing (s-MPS)
Z-score

14. 0.3 million (SD 88,000) reads 2.3 million (SD 474,000) reads
Shotgun Massively Parallel Sequencing (s-MPS)
Importance of read depth

15. Shotgun Massively Parallel Sequencing (s-MPS)
Importance of read depth

16. • Chr13 and Chr18 have relatively lower average GC content than Chr21
which has a modest average GC content.
chr13 chr18
SBS
SBL
chr21
1
1
5.8X 2.4X
5.7X 1.8X
• Coefficient of variation (CV) - Genomic representations of chr13 and chr18.
Z-score with GC bias correction
Shotgun Massively Parallel Sequencing (s-MPS)

17. Z-score with GC bias correction
Shotgun Massively Parallel Sequencing (s-MPS)

18. Trisomy 18
Detection rate
False positive rate
Trisomy 13Trisomy 21
100% (59/59)
0.3% (5/1,688)
91.7% (11/12)
0.9% (16/1,688)
99.1% (210/212)
0.1% (1/1,688)
Z-score with GC bias correction
Shotgun Massively Parallel Sequencing (s-MPS)

19. The count of mapped sites for a given chromosome of interest is normalized to counts
observed on another pre-determined chromosome (or set of chromosomes).
share similar GC content
Normalized Chromosome Value (NCV)
Shotgun Massively Parallel Sequencing (s-MPS)

20. Chr21/Chr1 Reference MeanChr 21/Chr1 Sample
Amy J. Sehnert, 2011
Chr21/Chr1 Reference SD
Normalized Chromosome Value (NCV)
Shotgun Massively Parallel Sequencing (s-MPS)

21.  Case control study including 532 pregnancies.
 Detection rate (Sensitivity) for T21 – 100% (89/89), No false-positive (404/404).
 2.6% unclassified (NCV between 2.5 and 4).
 Detection rate (Sensitivity) for T13 – 78.6% (11/14), No false-positive (485/485).
 Detection rate (Sensitivity) for 45,X – 93.8% (15/16), Specificity – 99.8% (416/417).
 Detection rate (Sensitivity) for T18 – 97.2% (35/36) , No false-positive (460/460).
Normalized Chromosome Value (NCV)
Shotgun Massively Parallel Sequencing (s-MPS)

22. Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm
Left probe Middle probe Right probe
Polymorphic site
Determine fetal fraction
Non-Polymorphic site
Determine copy number

23. Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm

24. Trisomic model
Disomic model
Observed proportion
for sample j
Estimated reference proportion
Fetal fraction
The FORTE algorithm estimates the risk of aneuploidy using an odds ratio comparing a model
assuming a disomic fetal chromosome and a model assuming a trisomic fetal chromosome.
Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm

25.  Case control study involving 400 pregnancies.
 Detection rate (Sensitivity) for T21 – 100% (50/50), specificity - 100% (297/297).
 1% test failure.
 Detection rate (Sensitivity) for T18 – 98% (49/50) , specificity - 100% (297/297).
Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm

26.  Case control study involving 3228 pregnancies.
 Detection rate (Sensitivity) for T21 – 100% (81/81), false-positive rate 0.03% (1/2888).
 4.6% test failure.
 Detection rate (Sensitivity) for T18 – 97% (37/38) , false-positive rate 0.07% (2/2888).
Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm

27. Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm, Microarray

28. 691 were disomic，
18 were trisomy 13，
37 were trisomy 18，
132 were trisomy 21.
Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm, Microarray

29. Targeted Massively Parallel Sequencing (t-MPS)
DANSE assay, FORTE algorithm, Microarray

30. parental genotypes
- Parental SupportTM
crossover frequency data (Hapmap Database)
billions of possible monosomic, disomic, and
trisomic fetal genotypes at measured loci fetal cfDNA fraction
expected sequencing data
bayesian statistics are used to determine the
relative likelihood of each hypothesis
observed Sequencing data
hypothesis with the maximum likelihood is
selected as the copy number and fetal fraction,
absolute likelihood of the call is the calculated
accuracy, analogous to a test-specific risk score.
multiplex PCR and NGS
- More than 11,000 SNPs covering chr 13,18,21,X,Y are
amplified by multi-plex PCR and sequenced by NGS
Read depth
Amplication bias
Noise parameters
Single nucleotide polymorphism (SNP)-based approaches

31. - Sensitivity and Specificity (145/166) - 100%
- 11/11 Trisomy 21
- 3/3 Trisomy 18
- 2/2 Trisomy 13
- 1/1 45,X
- 2/2 47,XXY
- Mean calculated accuracy - 99.9%
- 21/166 (12.7%) samples failed DNA quality test
Single nucleotide polymorphism (SNP)-based approaches

32. - 25/25 Trisomy 21
- 3/3 Trisomy 18
- 1/1 Trisomy 13
- 2/2 45,X
- 1/1 Triploidy
- Sensitivity and Specificity (229/242) - 100%
- Mean calculated accuracy - 99.9%
- 13/242 (5.37%) samples failed DNA quality test
Single nucleotide polymorphism (SNP)-based approaches

34. A binary hypothesis t-test and logarithmic likelihood ratio
L-score
Detection Rate False Positive Rate
T21
T18
100% (139/139)
100% (41/41)
0.03% (1/2820)
0.03% (1/2820)
Shotgun Massively Parallel Sequencing (s-MPS)

35. Z-score, Z-score with GC correction, Z-score with internal
reference chromosome
Shotgun Massively Parallel Sequencing (s-MPS)

36. Z-score with GC correction
Shotgun Massively Parallel Sequencing (s-MPS)

37. Z-score with GC correction
Shotgun Massively Parallel Sequencing (s-MPS)

38. Models for Incorporation of NIPT into Clinical Practice
False positive decrease
False negative rate
wont change
‘atypical’ chromosomal
abnormalities will be
missed

39. 10 weeks
12 weeks
Improved overall sensitivity Reduced invasive testing rate
false negative false positive
expensive
Models for Incorporation of NIPT into Clinical Practice

40. Increase detection rate to some extent
Reduce invasive testing rate to some extent
Models for Incorporation of NIPT into Clinical Practice

41. End

42. NIPT (Non-Invasive Prenatal Testing)
Digital PCR