1. Clinical Applications of NGS
- Genomics, NIFTY and Beyond
Dr. Ying(Abby) LIU, MD PhD
liuying2@genomics.cn

2. The stories all began
with these images!

3. Principle of High Throughput Sequencing
1. incorporation 1. Incorporation 1. Incorporation
2. Elute and scan 2. Elute and scan
3. Cleavage
C C
C C
T T
A A
G G
3

4. BGI Brief
• Largest genomics research center in the world
– 137 Hiseq2000, 27 AB Solid, 1 Roche 454, 2 Miseq, 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/ …

5. BGI Introduction
• Founded in 1999, for 1% of HGP
• Now the largest genomic organization in the world
9.1.1999 in London

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

7. The Only Gene Giant Covering 55 Countries
(Science 2.2012)
Copenhagen
Kobe
Philadelphia
UCD
Shenzhen
SA
Canberra

8. Partners
3000＋ Institutes/Hospitals，15000＋ Collaborators

10. Selected Large-Scale Projects in BGI

11. Selected Top Publications of BGI

12. Discovery Based on Large Data
1977
1980
1996 1998
1998 2000 2001 2002 2004 2005 2006 2007 2008 2009 2010 2011
2001
1.Life periodic table can be summarized
2.It will have laws as genetic central dogma
3. It can be elaborated in mathematical language
15

13. Organization Structure

14. BGI’sGene Test Strategy for Hereditary Diseases
Before Before
Prenatal Newborn
Marriage Pregnancy
Screening/ Screening/
Screening Screening
Diagnosis Diagnosis
Monogenic disease
Monogenic disease Monogenic disease
NIFTY
Hearing impairment
Hearing impairment Hearing impairment Monogenic disease Inherited metabolic
Hearing impairment diseases

15. NIFTY- Non-Invasive Fetal Trisomy Test
-BGI Experience

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

17. A safe prenatal screening test for
fetal chromosomal aneuploidy
Screening tests Diagnostic tests
• Serum biochemical test; • Karyotyping (G-
ultrasound scan banding or FISH)
• Non-invasive • Invasive
• Affordable • Expensive
• Less accurate • Highly accurate
• Low detection rate • High detection rate
• High false positive rate • Low false positive rate

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

19. 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

20. The Principle of NIFTY
cell-free DNA
50bp
Chr6
Chr18
ChrX
AACGT------ Chr21
CGATA------- Bioinformatics Chr7 Sequencing &
TAGGC------ Chr13
ATCGC------ Chr11 Alignment
ChrY
Normal
Sequence
Counting
T 21
Cov-chrN = Effective number of sequencing fragments on chr N of sample Bioinformatics
Effective number of sequencing fragments on chr N of reference sequence Analysis
ChrN t-Score Cov-Depth—Cov-Depth of reference sample
=
Of testing sample Standard deviation of reference sample
log ( Possibility of normal)
Report
L-value=
log (Possibility of abnormal)
26

21. The Statistic Model of NIFTY
100 cell-equivalent free DNA /ml plasma
95from mother, 5 from fetus
Normal Pregnancy T21 Pregnancy
Mother Chr 21: Fetus Chr 21: Mother Chr 21: Fetus Chr 21:
95X2=190 5X2=10 95X2=190 5X3=15
In maternal blood In maternal blood
Total Chr 21=190+10=200 Total Chr 21=190+15=205
Statistics for Tscore and L score
• GC correlation
• Binary hypothesis statistics analysis

22. The Performance Comparison of Statistics-I
Our approach with GC
Standard z-score approach z-score approach with GC- correlation and binary
bias removal hypothesis t-test
Items
Diagnostic
(number of cases) Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%)
Trisomy 13 detection
(2 cases) 50% 99.89 100% 100% 100% 100%
Autosomes Trisomy 18
detection(12 cases) 91.67% 100% 100% 99.89% 100% 99.96%
Trisomy 21 detection
(16 cases) 93.75% 100% 100% 100% 100% 100%
XO detection (3 XO,1 not not not not
XO/XX chimera) 75% 99.5%
available available available available
Heterosomes not not not not
XXY detection (1 case) 100% 100%
available available available available
not not not not
XXY detection (2 cases) 100% 100%
available available available available
Binary hypothesis not available not available available
Note: Data from 903 samples with full karyotypinginformation
28

23. The Performance Comparison of Statistics-II
Comparison of BGI’s approach (green)
with other approaches (orange and yellow)
PCT/CN2011/001070
NONINVASIVE DETECTION OF FETAL GENETIC ABNORMALITY

24. Clinical Validation (Phase I)
Study Design and Aim: – In 3464 Samples
3464 singleton pregnancies – Validation of the NIFTY in predicting
with high risk of T21, T18, T13
the fetal risk of trisomy 21, 18, and 13
in high risk population by a double
Plasma DNA from maternal
peripheral blood blind test
– Evaluation of sensitivity and specificity
NIFTY test
of the NIFTY by comparing to
karyotyping result (clinical gold
Double blind standard)
Karyotyping by AF, CVS, Cord
Bioinformatics analysis
blood
– Provide supports for large-scale test in
Calculate sensitivity and real clinical setting
specificity

25. NIFTY Validation Results (Phase I)
T21 T18 T13
NIFTY positive 189 64 10
Karyotyping positive 188 63 10
False positive No. 1* 1* 0
False Negative No. 0 0 0
Sensitivity 100.00% 100.00% 100.00%
Specificity 99.97% 99.97% 100%
False Positive Rate 0.03% 0.029% 0.00%
False Negative Rate 0.00% 0.00% 0.00%
Positive Predictive Rate 99.49% 98.44% 100.00%
*caused by insufficient sequencing depth

26. Large-scale Clinical Test (Phase II)
Qualified maternal blood samples (n=11184)
• 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 0.7% of all samples
Unable to produce results (n=79; failed DNA
extraction, library construction, or sequencing)
99.3% of all samples
Pregnant women with NIFTY results (n=11105)
Positive (n=190) Negative (n=10915)
Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8

27. NIFTY Results in 11105 Samples
Positive Results Negative
No. of Cases
T21 T18 T13 Results
NIFTY 11,105 140 42 0 10915
Karyotyping 182 + 2818 139 41 0 2818
False Positive Rate 0.03% 0.03% n/a
False Negative Rate 0% 0% n/a
Detection Rate 100% 100% n/a
Specificity 99.6% 99.6% n/a
Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8

28. Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8
Real Clinical Setting in 11,105 Singleton Pregnancies
Maternal age
Median (yr) 31
Advanced maternal age (>=35 yr) 3,858(34.74%)
18–24 yr (n, %) 918(8.27%)
25–29 yr (n, %) 3,439(30.97%)
30–34 yr (n, %) 2,890(26.02%)
35–39 yr (n, %) 3,188(28.71%)
≥40 yr (n, %) 670(6.03%)
Parity
Nullipara (n) 6,053/11,105(54.51%)
Gestational age at blood sampling
Median (wk) 20
Range (wk) 9-28
9 to 12 week (n, %) 371(3.34%)
13 to 16 week (n, %) 1,963(17.68%)
17 to 20 week (n, %) 5,598(50.41%)
21 to 24 week (n, %) 2,665(24.00%)
25 to 28 week (n, %) 508(4.57%)
History or family history of aneuploidies (n) 68/11,105(0.61%)
Presence of sonographic markers of chr abnormalities (n) 317/11,105(2.85%)
Had conventional Down syndrome screening tests
Yes – Screening positive (n, %) 4,522/6,948(65.08%)
Yes – Screening negative (n, %) 2,426/6,948(34.92%)
No – with one or more of other risk factors* (n, %) 2,770/11,105(24.94%)
No – without any risk factors* (n, %) 1,387/11,105(12.48%)

29. Current Clinical Pipeline
AMA,
Previous affected fetus,
Recurrent miscarriage,
Aneuploidy background
Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8

30. NIFTY Clinical Pipeline
>98% of invasive Advanced Maternal Age,
procedures could be avoided Previous affected fetus,
Recurrent miscarriage,
Aneuploidy background
Dan, S., et al., Prenatal Diagnosis, 2012: p. 1-8

31. Data Summary（Jan 2013 updated）
Testing Results
Phases Total cases
T21 T18 T13
Up-dated Multi-centered Clinical test of NIFTY 90847 724 253 65
(including the data of Phase I& II validation)
Subtotal for Phase I: Clinical Trial Study (2009-2010)
3464 189 64 10
Detection Rate >99.9% >99.9% >99.9%
False Positive Rate 0.0121% 0.0080% 0.0040%
PPV 99.38% 98.67% 96.43%
False Negative Rate 0% 0% 0%
NPV 100% 100% 100%

32. Other Rare Aneuploidies-II
T9
Sample ID: PDB11AJ00026
Age: 41
NIFTY: T9
Karyotyping: T9

33. Other Rare Aneuploidies-III
T16
Sample ID:
PDB11AJ00783
Age: 30
NIFTY: T16
FISH: T16

34. Other Rare Aneuploidies-IV
Mosaic T21
Sample ID: PDB12AO00267
Age: 38
NIFTY: T21
Karyotyping: 47, XX, +21(88%)

35. Other Rare Aneuploidies-IV
T21-T7-XXY complex placental mosaic
Sample ID: PDB12AL00732
Age: 37
NIFTY: T21-T7-XXY/XY
Karyotyping: CVS T21-T7-XXY/XY; AF euploid

36. NIFTY is Suitable for
Gestational
age 12-24
weeks
Recurrent
AMA or high
miscarriage or
risk
IVF
NIFTY
Refuse to
Inappropriate
receive invasive
for invasive test
test

37. NIFTY Workflow
Hospital
Pre-test
Blood Bar- Plasma Post-test
counselin preparation
collection coding counseling
g
BGI Clinical Laboratories: 10 days
Laboratory Bioinformatics Report
testing analysis delivery

38. BGI Systems
Sample Management Laboratory
Effective lab space
separation
Unique Identification
Progressively
decreased pressure
Sample Location
Restricted traffic flow
Storage Capability
46

39. Sample Requirements
Blood sampling Packing for delivery
• 5-10ml in EDTA tube • Strong support in case of
• Clearly labeled damage
• Enough dry ice to keep
frozen
Plasma preparation Sample storage
• Immediately extract • -20℃ for a week, -80℃ for
plasma long term
• Stored at 4℃, extract in 8 • Avoid room temperature
hours and repeated thaw

40. NIFTY 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, T18, or T13
-Diagnostic procedure such as amniocentesis or
cordocentesis is recommended
More than 98% of cases

41. Other Rare Aneuploidies
Chromosomal number variations (CNVs) on other
autosomal chromosomes
Chromosomal number variations (CNVs) on sex
chromosomes
Results of other prenatal tests such as biochemical and
ultrasound tests should be considered
Diagnostic procedure such as amniocentesis/cordocentesis
is suggested

42. BGI Papers
52

43. NIFTY Summary
SA F E 5mlmaternal blood
Sensitivity99.9%
AC CU R A T E Specificity99.91%
FA S T 10working days
EA R L Y from12thweek on
54

44. Monogenic Diseases Test
-BGI Experience

45. Background
• a single mutated gene, Mendelian
pattern of inheritance Autosomal dominant
Autosomal recessive
• point mutation, frame shift, deletion, X-linkage dominant
insertion X-linkage recessive
Y-linkage
• Autosomal dominant/recessive; X-
linked dominant/recessive; Y-linked
• >6700 monogenic diseases，10-50
more every year.
• 1 in every 200 newborns
• Genetic testing is the gold standard

46. Monogenic Disease Strategies at BGI
Target region capture combined with NGS
Exome/whole genome resequencing
Sanger sequencing

47. Target Sequence Capture + NGS
Target region
PCR, capture,
Sanger NGS
sequencing

48. Mutation Databases
dbSNP Hapmap 1000 genome Local genome
frequency frequency project database
Normal persons SNV
database
Single-gene disease
variants database
59

49. Advantages
High throughput
• Multiplex testing
Full coverage
• Simultaneously detect point mutation, minor insertion/deletion
(<20bp), and large homozygous deletion/duplication
Accurate
• Test region covers >95% target gene, sensitivity >99%
Automated bioinformatics analysis
• Fast identify mutation; completed disease mutation database
and control genetic polymorphism database available

50. Monogenic Diseases
Psychological health & behavior
9 diseases Visual system
Brain & Nervous system 16 diseases
1 disease Ear, Nose & Throat 6 diseases
Oral & dental
1 disease Immune system 6 diseases
Congenital tumor
Respiratory system 2 diseases
Circulatory system
54 diseases Endocrine &
Metabolic system Digestive system 7 diseases
8 diseases Multi-system Blood & lymphocyte system 6 diseases
Urinary & reproductive system
5 diseases
Musculoskeletal &
25 diseases Connective tissue system
Skin, Hair & Nails 7 diseases
160 common monogenic diseases across 16physiologicaland functional systems

51. List of Monogenic Diseases (160 diseases)
Disease Gene and mutation
INPP5E，TMEM216，TMEM67，OFD1，NPHP1，CC2D2A，CEP290，ARL13B，
Joubert syndrome
Disease AHI1，RPGRIP1L Gene and mutation
Spastic Paraplegia, Autosomal Recessive and X-linked
Alport Syndrome, X-Linked CYP7B1，SPG7，SPG11，ZFYVE26，SPG20，SPG21，L1CAM，PLP1，SLC16A2
COL4A3，COL4A4，COL4A5
Disease
Spastic Paraplegia, Autosomal Dominant dominant
Polycystic Kidney Disease, Autosomal PKD1，PKD2 Gene and mutation
ATL1，SPAST，NIPA1，KIAA0196，KIF5A，HSPD1，BSCL2，REEP1，ZFYVE27
Polycystic KidneyIIDisease, Autosomal Recessive
Citrullinemia, Type
Metachromatic Leukodystrophy PKHD1
ASS1，SLC25A13，ASL
ARSA
CA4，CRX，FSCN2，IMPDH1，NRL，PRPF3，PRPF31，PRPF8，
ACADVL
RetinitisDehydrogenase,Form Long-Chain, Deficiency Of
Acyl-Coa Pigmentosa, Very
Refsum Disease, Infantileautosomal dominant PEX1，PEX2，PEX26
Acyl-Coa Dehydrogenase, Short-Chain, Deficiency Of
Arts syndrome PRPH2，RHO，ROM1，RP1，RP9
ACADS
PRPS1
Acyl-Coa Dehydrogenase, Medium-Chain, Deficiency Of
Agenesis Of The Corpus Callosum With Peripheral Neuropathy
ABCA4，BEST1，CERKL，CNGA1，CNGB1，CRB1，EYS，IDH3B，
ACADM
SLC12A6
Argininosuccinic Aciduria KLHL7，LRAT，MERTK，NR2E3，PDE6A，PDE6B，PRCD，PROM1，
ASL
Peutz-Jeghers Syndrome autosomal recessive and X-link recessive
Retinitis Pigmentosa, STK11
Argininemia PRPH2，RGR，RLBP1，RP2，RPE65，RPGR，SAG，SEMA4A，
ARG1
Adenomatous Polyposis Of The Colon APC
Aspartylglucosaminuria SNRNP200，TOPORS，TULP1，USH2A，RHO，NRL
AGA
Gilbert syndrome UGT1A1
Usher Syndrome to cystathionine beta-synthase deficiency
Homocystinuria due USH2A，CDH23，CLRN1，GPR98
CBS
Cystic Fibrosis CFTR
Waardenburg syndrome
Tyrosinemia Type I
Tuberous Sclerosis
EDNRB，MITF，PAX3
FAH
TSC1，TSC2
Glutaric Acidemia I
Osteogenesis Imperfecta GCDH
COL1A1，COL1A2，CRTAP
Marfan Syndrome FBN1
IsovalericAcidemia
Osteopetrosis IVD
CLCN7
Bloom Syndrome BLM
Fabry Disease
Amyotrophic Lateral Sclerosis GLA
SOD1，FUS，SETX，TARDBP，ANG，VAPB，FIG4，ALS2
ACTG1，CCDC50，COCH，DFNA5，DIAPH1，DSPP，EYA4，GJB2，GJB3，GJB6
Krabbe Disease GALC
Nonsyndromic Deafness,Duchenne Dominant (including mitochondiral
Muscular Dystrophy, Autosomal Type/Becker Type DMD
，GRHL2，KCNQ4，MIR96，MYH14，MYH9，MYO1A，MYO6，MYO7A，
Niemann-Pick Disease NPC1，NPC2，SMPD1
deafness) Congenita, Autosomal Dominant /Amyotonia Congenita
Myotonia POU4F3，SIX1，SLC17A8，TECTA，TMC1，WFS1，m.1494C>T，m.1555A>G，
CLCN1
AGL，ALDOA，ENO3，G6PC，GAA，GYS1，LDHA，PYGM，PGM1，
m.7445A>G，m.7510T>C，m.7472insC，m.7511T>C
Glycogen Storage Disease
Epiphyseal Dysplasia, Multiple,Autosomal Recessive SLC26A2
PHKG2，GBE1
Galactosemia Permanent Neonatal CDH23，DFNB31，DFNB59，ESPN，ESRRB，GJB2，GJB6，GRXCR1，HGF，
GALT
Diabetes Mellitus, GCK，KCNJ11，INS，ABCC8，PDX1，PTF1A
LOXHD1，MYO15A，MYO3A，MYO6，MYO7A，OTOA，OTOF，PCDH15，RDX
Nonsyndromic Deafness,Type II Nongoitrous
Hypothyroidism, Congenital, Recessive (including mitochondiral
Mucopolysaccharidosis Autosomal TSHR，TSHB，NKX2-5
IDS
，STRC，TMIE，TMPRSS3，TRIOBP，TMC1，USH1C，SLC26A4，BSND，
Phenylketonuria Dystrophica, Autosomal Dominant
deafness)
Epidermolysis Bullosa PAH
COL7A1
PTPRQ，LHFPL5，LRTOMT，SLC26A5，MARVELD2，CLDN14，TPRN，LHFPL5，
congenital Dysplasia,cortical hyperplasia
Ectodermal adrenal Hidrotic, Autosomal Dominant CYP21A2
GJB6
m.1494C>T，m.1555A>G，m.7445A>G，m.7510T>C，m.7472insC，m.7511T>C
Maple Syrup Urine Disease
Deafness, X-linked Recessive BCKDHA，BCKDHB，DBT
FANCA，FANCB，FANCC，BRCA2，FANCD2，FANCE，FANCF，FANCG，
POU3F4，PRPS1
Fanconi Anemia
Wilson Disease ATP7B
FANCI，BRIP1，FANCL，FANCM，PALB2，RAD51C，SLX4
Ataxia-Telangiectasia ATM
Multiple Endocrine Neoplasia RET
Neurofibromatosis NF1，NF2
Exostoses, Multiple EXT1，EXT2
Hereditary Breast and Ovarian Cancer BRCA1，BRCA2

52. Workflow
Pre/posttest Sample DNA Library
counseling collection extraction construction
Report Family Bioinformatic
Sequencing
delivery validation analysis
Turnaround time: 35 working days

53. Data analysis
• Sequencing quality assessment (depth, coverage)
• Variation sites (dbSNP frequency, HapMap frequency,
1000 genome frequency, local database frequency)
• Possible mutations (nucleotide change – amino acid
change)
• Validation & confirmation of pathogenic gene

54. Report Template

55. This Test is Recommended for …
Clinics:
• Patients with clear phenotypes
• Patients with suspected disease
• People with no phenotypes but have family history
• Carrier screening
Research:
• Assisting diagnosis: provide genetic proof for
uncertain phenotypes
• Disease research: deeper understanding of genetics,
human mutations

56. Monogenic Disease Gene Detection
• 160 Monogenic diseases across 16 physiological and
functional systems
• Peripheral blood or genomic DNA
• Turnaround time: 35 working days
• Available at all life stages – life cycle solution
Before Before
Prenatal Neonatal
marriage pregnancy

57. And Beyond…
• Genetic testing
– Thalassemia&hemopathy
– HPV, HCV, HBV
– Cancer
– Metabolic Disorders
– Hearing Impairment
– Microdeletion&Microduplication
– PGD
– Arrhythmia
– High resolution HLA typing
– Exome Sequencing
– Personal Genome
– …

58. Clinically Affordable Soon
$1000 genome will be true very soon

59. Recent Promotions
• NIFTY (3.18.2013 - 3.29.2013)
+ FREE Test for Newborn Metabolic Disorders
+ FREE Test for Hearing Loss
• HPV Testing (3.18.2013 - 4.18.2013)

60. Life Cycle Solution
&
Grand Scale Collaboration
www.genomics.cn http://birth.bgi-health.com/index

61. More information:
www.genomics.cn or http://birth.bgi-health.com/index