SANGER SEQUENCING - 1st generation sequencing Sanger Sequencing Workflow: PCR amplification (target enrichment) PCR purification (primer, dNTPs) Sequencing reaction (bi-directional) Sequencing purification (primer, dNTPs, ddNTPs) Electrophoretic run on sequencer Sequencing lecture Alignment to reference SANGER SEQUENCING: LIMITATIONS Analytical sensitivity*: 99% PCR-Based  no detection deletion/duplication rearrangements del/dup BRCA = 4-28% of all BRCA mutations in most population** Level of mosaicism > 20% Low throughput (82496 capillary tubes) Labor intensive Time consuming High cost (large size gene or more genes)

1. Dal Sanger al NGS nello studio delleDal Sanger al NGS nello studio delle
mutazioni BRCAmutazioni BRCA
Simona De Toffol,Simona De Toffol, TOMA Advanced Biomedical Assays S.p.A.TOMA Advanced Biomedical Assays S.p.A.
sdetoffol@tomalab1.comsdetoffol@tomalab1.com
PRIMO INCONTRO DI GENETICA ONCOLOGICAPRIMO INCONTRO DI GENETICA ONCOLOGICA
19 Febbraio 201619 Febbraio 2016

2. BRCA 1 e BRCA2BRCA 1 e BRCA2
BRCA1: 1731
BRCA1
BRCA2
BRCA2: 2000

3. SANGER SEQUENCING -SANGER SEQUENCING - 1st
generation sequencing
For the past 30 years until now, the Sanger method has been the gold standard for DNA sequencing
- Chain termination sequencing
Sanger Sequencing Workflow:
 PCR amplification (target enrichment)
 PCR purification (primer, dNTPs)
 Sequencing reaction (bi-directional)
 Sequencing purification (primer, dNTPs,
ddNTPs)
 Electrophoretic run on sequencer
Sequencing lecture
 Alignment to reference
1977 (Sanger et al.) 1987 (Prober et al.)

4. SANGER SEQUENCING: quality scoreSANGER SEQUENCING: quality score
Phred quality score: quantifies the base-calling
error probability: Q= -10log10 p
Analytical sensitivity: 99%

5. SANGER SEQUENCING: LIMITATIONSSANGER SEQUENCING: LIMITATIONS
Analytical sensitivity*: 99%
PCR-Based  no detection deletion/duplication rearrangements
del/dup BRCA = 4-28% of all BRCA mutations in most population**
Level of mosaicism > 20%
Low throughput (82496 capillary tubes)
Labor intensive
Time consuming
High cost (large size gene or more genes)
the gold standard for BRCA1-BRCA2 variants identification  SEQUENCING + MLPA
*Ewing et al, Genome Research 1998. **Kwong et al, Cancer Genet 2015.

6. FROM SANGER to NGS SEQUENCINGFROM SANGER to NGS SEQUENCING
2001
1990
13-years  $2.7 billion
2-months< $1 million
$1000 genome programme
2003
NHGRI began to fund basic and industrial research to
develop new methods of sequencing for commercial use
Hayden EC. Nature, 2014, 507:294-5 – Wheeler et al, Nature, 2008.

7. Next Generation SEQUENCINGNext Generation SEQUENCING
2005: the first NGS platform (454 GS20 sequencer – Life Sciences - Roche)
NGS: sequencing of spatially separated, clonally amplified DNA templates or
single DNA molecules performed in a flow cell in a massive parallel manner
2nd
generation: clonally amplified
DNA or sequence/exon capture
3rd
generation:
single molecule DNA
1st
generation

8. Next Generation SEQUENCING: ApplicationsNext Generation SEQUENCING: Applications
Clinical genomics
Prenatal diagnosis (NIPT)
Cancer genomics
Forensic genomics
Metagenomics
(microbiome – infectious agents)
Drug resistance
Pharmacogenomics
Pharmacogenetics
Epigenomics
Trascriptomics
ChIP seq
Agrigenomics
Whole Genome Sequencing (WGS):
characterize entire genomes of any
size and complexity
Exome Sequencing :
sequence protein coding regions, as
cost-effectivealternative to WGS
Targeted Resequencing:
sequence specific genes or other
regions of interest
De novo Sequencing:
sequence and assemble novel
genomes
Kahvejian et al. Nature Biotechnology 2008

9. Next Generation SEQUENCINGNext Generation SEQUENCING
2st
generation sequencing
 enrichment on target: clonally amplified DNA or sequence /exon capture
 library preparation: adapter sequences complementary to platform
 barcoding: molecular tagging with unique sequence-based codes (pooling)
 sequencing by synthesis (Illumina, IonTorrent)
 sequencing by ligation (Solid)
 resulting sequence reads are processed through a computational pipeline
PacBio $ 700K
3rd
generation sequencing
MinION PromethION/GridION

10. NGS - 2NGS - 2ndnd
generationgeneration
Sequencing by reversible dye terminators
1998 Solexa - 2006 Illumina
Main bench-top sequencers (Illumina-Life Technologies) > 85% market
2011

11. COMPARISON OF NGS SYSTEMSCOMPARISON OF NGS SYSTEMS
Each sequencing platform has advantages and disadvantages
 Lower error rate
 Lowest cost per base
 Wide range of applications
 Short read length (50-150bp)
 Runs take multiple days
 No de-novo assembly
 Low error rate
 Medium/low cost per base
 Fast run (hours)
 Low startup costs
 No amplification required
 Extremely long read
lengths (max 15000bp)
 de-novo assembly
 High error rates (5-15%)
 Medium/high cost per base
 High startup costs
no mutation detection (diagnostic)
2012
2014
2012
 Homopolymers reads problem
 Read lengths only 100-200bp
 coverage bias with GC-rich regions
 New, developing technology

12. COMPARISON OF NGS SYSTEMCOMPARISON OF NGS SYSTEM
Differences in sequencing chemistry of each platform result in differences in total
sequence capacity, sequence read length, sequence run time and
final quality and accuracy of the data
https://en.wikipedia.org/wiki/DNA_sequencing

13. NGS: ERROR RATE AND ACCURACYNGS: ERROR RATE AND ACCURACY
Quality string= Phred –like quality scores
Depth of Coverage:
the average number of times a given region has been sequenced by indipendent reads
Base-calling error probability:
The accuracy of
variant calling depends
on the depth of
sequence coverage
and improves with
increasing coverage.

14. NGS: ERROR RATE AND ACCURACYNGS: ERROR RATE AND ACCURACY
Base calling
Read alignment
variant calling
variant annotation
Instrument (chemistry)
dipendent  FastQ files
on reference sequence
(BWA, Botwie2, Picard)
Detection of the DNA variants in
the sequence analyzed as
compared with a reference
sequence (GATK Tools)
Assigning functional
information to DNA
variants
Data analysis and data storage BigData
Data analysis: bioinformatic pipeline
Data analysis accuracy?
BAM SAM files
VCF files
Cloud computing solution:
“on demand” access to
supercomputing

15. NGS IN CLINICAL LABs: VALIDATIONNGS IN CLINICAL LABs: VALIDATION
2013 2014 (update 2012 CMGS) 2016
TEST/PIPELINE
Validation
Analytical Sensitivity
Analytical SpecificityPositive samples: different variant
types (in/dels, substitutions, CNVs)
Library preparation  “index tagging” (barcoding)
Quality scores reporting suggested (Q30- data analysis versioning)
Region of interest (coding regions and splice-sites) –”horizontal coverage”
“vertical coverage” (analysis resolution)  low coverage (Sanger)
confirmation (Sanger or other technologies)
limits (CNVs, indels, homopolymers, allelic dropout
pseudogenes, highly GC rich regions, repetitive elements)
filtering of variants (exome-genome sequencing)
data storage (vcf file)
Incidental findings (WES or WGS)
low coverage increases
the risks of missing
variants (FNs) and
assigning incorrect allelic
states (zigosity), especially
in the presence of
amplification bias, and
decrease the ability to
effectively filter out
sequencing artifacts,
leading to FPs
For clinical molecular genetic test validation: Mattocks et al, Eur J Hum Genet 2010:18:1276-1288

16. NGS: BRCA1-BRCA2 genesNGS: BRCA1-BRCA2 genes
Validation:
 MiSeq/MiSeq Reporter Software no indels > 9bp
 MiSeq/QSAP
 PGM/Torrent Suite
521 clinical samples
 MiSeq/QSAP 100% sensitivity
 PGM/Torrent Suite no 2 ins/del cases
(10bp-64bp)
2015 2015
2012
100% sensitivity
(379 BRCA1/BRCA2 variants)
2015

17. NGS: BRCA1-BRCA2 genesNGS: BRCA1-BRCA2 genes
GeneReadTM
DNAseq
BRCA1 and BRCA2 panel
CE-IVD 2012
BRCA1 and BRCA2
2015: The Gene Reader TM
(only clinical use)
Buccal swap at home
Report in 2 weeks
CE-IVD (520€)
SeqNextTM
Software
DropGenTM
Software

18. NGS: BRCA1-BRCA2 genes and more…NGS: BRCA1-BRCA2 genes and more…
familial breast cancer
moderate and low-risk genes:
ATM, CHECK2, BRP1, PALB2
MLH1, MSH2, MSH6, PMS2,
EPCAM, MUTYH, BARD1,MRE11A,
NBN, RAD50, RAD51C,RAD51
Predisposition to Breast/Ovarian cancer:
high-risk genes (~25%):
BRCA1-BRCA2
PTEN-CDH1-STK11-P53
2013
Shiovit S & Korde LA, Annals of Oncology, 2015. 26:1291-1299
Unquantified or variable (SNPs):

19. NGS: BREAST/OVARIAN CANCER PANELSNGS: BREAST/OVARIAN CANCER PANELS

20. NGS: HEREDITARY CANCER PANELSNGS: HEREDITARY CANCER PANELS

21. NGS in clinical settings: advantagesNGS in clinical settings: advantages
High analytical sensitivity (validated!!!) – Targeted sequencing >99.9% – Q ≥ 30
Identification of deletion/duplication rearrangments (CNVs)  no MLPA
Identification of variants in regions not typically tested (deep intronic, promoter)
High troughtput (massive parallel sequencing)
Low cost Reduced tournaround time  a significant impact
in clinical practice
High resolution/coverage (detecting mutant allele as low >5%)  Mosaicism
Tumor tissue
Multi-gene panels: genes can be tested simultaneously rather than sequentially
(low cost, reduced time  a significant impact in clinical practice)
Clinically use of WES/WGS  Personalized medicine

22. NGS in clinical settings: challengesNGS in clinical settings: challenges
Clinical validation of NGS  High confident data analysis/interpretation
(VEQ)
“It may be necessary that the maximum
benefit of this new technology is translated
into optimal patient care”
New clinical
work-flow?
Clinically use of WES/WGS  Incidental findings
Pilgrim et al , BJS 2014 - Shiovit z & Korde , Annals of Oncology, 2015.
New selection criteria for BRCA testing?
Hereditary Panels (Breast/Ovarian Cancer):
 Which genes? Clinical utility and management for moderate-low risk genes variant detection?
 Variants with Unknown Significance (VUS)? Multiple variants?
New sophisticated computerized alghoritm
to estimate risk calculation

23. VARIANT INTERPRETATIONVARIANT INTERPRETATION
2008
http://enigmaconsortium.org
2015
2011
2009 - ENIGMA:
Multidisciplinary
International Consortium
for classification of
BRCA1-BRCA2 variants

24. Thanks for your attentionThanks for your attention
Prof. GiuseppeProf. Giuseppe
SimoniSimoni
Dott. FedericoDott. Federico
MaggiMaggi
Lab.TOMALab.TOMA
Dott.ssa FrancescaDott.ssa Francesca
Romana GratiRomana Grati
Dott.ssa Anna Maria RuggeriDott.ssa Anna Maria Ruggeri
Dott.ssa Francesca DulcettiDott.ssa Francesca Dulcetti
Dott.ssa Elisa GaetaniDott.ssa Elisa Gaetani
Dott.ssa Elena NiadaDott.ssa Elena Niada
Dott.ssa Livia MarcatoDott.ssa Livia Marcato