1. Non-invasive Prenatal Trisomy test Leon Liang BGI Health Europe A safe prenatal testing for fetal chromosomal aneuploidy

2. Common fetal aneuploidy Trisomy 21 (1/700-750) 80% reach to adulthood Down syndrome Trisomy 18 (1/6000) <10% reach to 1 year old Edwards syndrome Trisomy 13 (1/10000-20000) Most die after birth Patau syndrome Others: Turner syndrome (XO), Klinefelter syndrome (XXY), triple X syndrome (XXX), etc.

3. Current screening & diagnostic tests * Data present in 5% false positive rate Fergal D, Jacob A, et al. The New England Journal of Medicine, 2005

4. Screening testsDiagnostic tests Serum biochemical test; ultrasound scan Non-invasive Cheap Less accurate Low detection rate High false positive rate Karyotyping (G-banding or FISH) Invasive Expensive Highly accurate High detection rate Low false positive rate

5. What ’ s NIFTY test? Non-Invasive Fetal TrisomY A superior screening test High detection rate and low false positive rate Non-invasive method based on NGS and bioinformatics Analysis of fetal cell free DNA in maternal plasma Evaluate the likelihood of fetal trisomy 21, 18, and 13

6. Features of NIFTY Accurate Safe Early Optimization Simple New detection technology based on NGS; Sensitivity and specificity > 99% No risks of intrauterine infection and miscarriage Fetal trisomy risk can be evaluated as early as 12 gestational week Reduce clinical pressure of unnecessary invasive tests Only 5ml of peripheral blood is needed; easy to handle in clinical practice

7. Scientific discovery Fundamental Features of Cell-Free Fetal DNA Short fragments of 145-200bp, derived from placental trophocytes 970 times greater than fetal cells DNA in maternal blood. Detectable in maternal plasma from the 5th week of gestation. Concentration increase as the gestation age grows Disappears soon after childbirth.

8. Sequencing revolution Sanger NGS

9. Principle of NIFTY Chr6 Chr18 ChrX Chr21 Chr7 Chr13 Chr11 ChrY ….. Plasma DNA Total cfDNA sequencing Reads count NormalT-21 Reads alignment Bioinformatics analysis

11. – Validation of the NIFTY in predicting the fetal risk of trisomy 21, 18, and 13 in high risk population by a double blind test – Evaluation of sensitivity and specificity of the NIFTY by comparing to karyotyping result (clinical gold standard) – provide supports for large-scale test in real clinical setting Clinical validation in 3464 samples Study design and aim:

12. NIFTY results in 3464 samples T21T18T13 NIFTY positive 1896410 Karyotyping positive 1886310 False positive No. 1*1* 1*0 False Negative No. 000 Sensitivity 100.00% Specificity 99.97% 100% False Positive Rate 0.03%0.029%0.00% False Negative Rate 0.00% Positive Predictive Rate 99.49%98.44%100.00% *caused by insufficient sequencing depth

13. Large scale clinical test (2009-2011) Qualified maternal blood samples (n=11184) Unable to produce results (n=79; failed DNA extraction, library construction, or sequencing) Pregnant women with NIFTY results (n=11105) Positive (n=190)Negative (n=10915) 0.7% of all samples 99.3% of all samples Gestational week from 9 – 28 weeks, averagely 20 weeks Maternal age from 18 – 45 years, averagely 31 years 4522 screening test high risk pregnancies 2426 screening test low risk pregnancies 2720 other high risk factors (AMA, abnormal NT, previous abnormal pregnancy, etc.) 1387 screening not done Shan Dan, et al., Prenatal Diagnosis, 2012: p. 1-8.

14. NIFTY results in 11105 samples No. of cases Positive Results Negative results T21T18T13 NIFTY11,10514042010915 Karyotyping182 + 28181394102818 False positive rate0.03% n/a False negative rate0% n/a Detection rate100% n/a Specificity99.96% n/a Shan Dan, et al., Prenatal Diagnosis, 2012: p. 1-8.

15. A T21 case was missed by biochemical screening Sample ID: PDP10003761 Age: 31 Screening test: 1/510 （ Low risk ） NIFTY: T21 Karyotyping: 47, XX, +21 15 Other rare aneuploidies

16. T9 Sample ID: PDB11AJ00026 Age: 41 NIFTY: T9 Karyotyping: T9

17. T16 Sample ID: PDB11AJ00783 Age: 30 NIFTY: T16 FISH: T16

18. Mosaic T21 Sample ID: PDB12AO00267 Age: 38 NIFTY: T21 Karyotyping: 47, XX, +21 (88%)

19. T21-T7-XXY complex placental mosaic Sample ID: PDB12AL00732 Age: 37 NIFTY: T21-T7-XXY/XY Karyotyping: CVS T21-T7-XXY/XY; AF euploid

20. NIFTY clinical pipeline AMA, Previous affected fetus, Recurrent miscarriage, Aneuploidy background

21. Test workflow Pre-test counseling Blood collection Bar-coding Plasma preparation Laboratory testing Bioinformatics analysis Report delivery Post-test counseling Hospital BGI Clinical Laboratories: 10 days

22. 22 Sample Management System Unique Identification Sample LocationStorage Capability Effective lab space separation Progressively decreased pressure Restricted traffic flow Laboratory System

24. NIFTY is not suitable for Detection of balanced rearrangements and low level of mosaicism The pregnant woman is a chromosomal aneuploidy carrier If either of the parent has chromosomal structural abnormalities e.g. duplication, deletion, translocation, etc. If the pregnant woman receives allogenic DNA importation prior to NIFTY e.g. blood transfusion, transplantation, stem cell therapy, etc.

25. Report Genetic testing report Low risk: the fetus is unlikely to be T21, T18, or T13. No special medical procedure is recommended. Routine prenatal checks is suggested. High risk: the fetus is likely to be T21, or T18, or T13. diagnostic procedure such as amniocentesis or cordocentesis is recommended. More than 98% of cases

26. Delay notification QC: Caused by either the experiment or blood sample quality; need to repeat the experiment Data fluctuation: Caused by high data deviation; need to repeat the experiment cfDNA concentration low: Need to repeat the experiment If repeating experiment still cannot solve the problem, blood re- sampling is required.

27. Re-sampling notification QC: Caused by poor blood sample quality cfDNA concentration low Previous NIFTY failed to produce informative result, and gestational age is more than 22 weeks

28. Sample requirement Blood sampling 5ml in EDTA tube Clearly labeled Plasma preparation Immediately extract plasma Stored at 4 ℃, extract in 4 hours

29. Packing for delivery Strong support in case of damage Enough dry ice to keep frozen Sample storage -20 ℃ for a week, -80 ℃ for long term Avoid room temperature and repeated thaw

30. BGI papers 30

33. More Choice NIFTY expressNIFTYNIFTY plus Detection of T21, T18, T13 Gender Twin pregnancies Sex chromosome abnormality Microdeletion & Microduplication Monogenic disease Turn around time6 days14 working days

34. NIFTY express Data Chr21 (61 T21 cases) Sensitivity96.7% (59/61) Specificity98.4% (1561/1586) Chr18 (16 T18 cases) Sensitivity100% (16/16) Specificity98.7% (1610/1631) Chr13 (13 T13 cases) Sensitivity92.3%(12/13) Specificity99.1%(1619/1634) Performed on Ion Proton platform, extremely fast Performed in Czech Republic BGI-GENNET co-lab. EU based service. Similar price Validated on 1647 samples

35. NIFTY plus Detection of Microdeletion syndromes Non-invasive Monogenic disease detection Results of other prenatal tests such as biochemical and ultrasound tests should be considered. Diagnostic procedure such as amniocentesis/cordocentesis is suggested.

36. Summary Non- invasive 5ml maternal blood No risk of intrauterine infection and miscarriage Accurate Sensitivity >99% Specificity >99% Turnaround time 10 days Early detection As early as 12 weeks

37. About BGI Largest genomics research center in the world – 137 Hiseq2000, 27 AB Solid, 1 Roche 454, 1 Ion Torrent, 30 AB 3730 Strong leader board and qualified employees – More than 4000 employees including 1500 bioinformaticians Extraordinary super computer and cutting-edge cloud computing technique – 102T flops/ 10PB storage/ 20TB memory Numerous high-quality publications on top academic Journals – Nature/ Science/ …

38. Selected Top Publications of BGI 38

39. Milestones of BGI BGI-HZ, 2001 BGI-Beijing, 1999 BGI-WH, 2010 BGI-SZ, 2007 BGI-HK, 2009 BGI-Americas, 2010 BGI-Europe, 2010

40. Platforms of BGI 40 Computing and bioinformatics platform Sequencing platform Agricultural genomics platform Cloning platform Healthcare platform Microbiology platform Proteomics platform

41. Research collaborators worldwide