Implementation of BRCA Oncomine panel Enrico Tagliafico · 2020. 6. 13. · A m pliSeq BRC A 1 and...

Tagliafico ESHG 2017.pptm 03/03/2017

European Human Genetics Conference MAY 27-30, 2017 – Copenhagen, Denmark

Implementation of BRCA Oncomine panel for germline and somatic variant analysis

Enrico Tagliafico MD, PhD, Modena, Italy

Center for Genome Research

University of Modena and Reggio Emilia, Italy

Clinical Genomics Laboratory

University Hospital, Modena, Italy

3.2%

Tagliafico ESHG 2017.pptm 03/03/2017 Introduction


The Lab

# of samples processed/year Oncology (NGS) 500

Rare Diseases (NGS) 300

Microbiology/Virology (Sanger) 1700

Cytogenomics (CMA) 250

Personnel Biologists / Biotechnologist 5

Bioinformaticians 1

Physicians 3

6.5%

Tagliafico ESHG 2017.pptm 03/03/2017 Introduction


The Lab

Major Equipment: NGS: Ion Torrent PGM Ion Torrent S5 Illumina MiSeq DX

Sanger: 3130xL Genetic Analyzer 3500xL Dx Genetic Analyzer

Cytogenomics: Affymetrix GeneChip System

Automation: IonChef 2x Hamilton Starlet 2x QIAgility

9.7%

Tagliafico ESHG 2017.pptm 03/03/2017 Workflow


Sample Library and

template preparation

Ion HiQTM Sequencing Data Analysis

FFPE & Blood Ion Chef TM or manual +

Ion One Touch

Ion PGMTM System

Ion ReporterTM Software

12.9%

Tagliafico ESHG 2017.pptm 03/03/2017 Ion BRCA Community panel vs Oncomine BRCA


Total sample size

Ion Ampliseq BRCA1 and BRCA2 Community panel (2014-2016)

Oncomine BRCA Research Panel (2017)

Manual Manual + Ion Chef Ion Chef Manual Manual +

Ion Chef Ion Chef

Germline analysis 832 6 -- -- 16 185

Somatic analysis -- -- -- -- -- 15

Tot samples 838 216

Ion Ampliseq BRCA1 and BRCA2 Community panel

Oncomine BRCA Research Panel

Amplicons 167 265

Pools 3 2

Library automation NO YES

Template automation YES YES

Panel Characteristics

16.1%

Tagliafico ESHG 2017.pptm 03/03/2017 Germline Analysis: Community Panel vs Oncomine


20X 50X 100X 250X 500X 750X 1000XBRCA1 and BRCA2Community Panel 97.9 93.2 86.7 75.7 64.9 59.1 55.2

Oncomine BRCAResearch Assay 99.9 99.8 99.2 94 77.2 50.3 23.6

0%

25%

50%

75%

100%

Ampl

icon

s (%

)

19.4%

Mean coverage depth=500X

Tagliafico ESHG 2017.pptm 03/03/2017 Germline Analysis: Community Panel vs Oncomine


50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

0 500 1000 1500 2000 2500 3000 3500 4000

Am

plico

ns

(%

)

Mean coverage (#reads)

Oncomine BRCA Research Panel (216 runs)

coverage >50X coverage >100X

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

0 500 1000 1500 2000 2500 3000 3500 4000

Am

plico

ns

( %

)

Mean Coverage (#reads)

BRCA1 and BRCA2 Community panel (838 runs)

coverage>50X coverage>100X

22.6%

Tagliafico ESHG 2017.pptm 03/03/2017 Oncomine BRCA performances upon different experimental settings


#samples/Chip 8/316 16/316 8/318 16/318 32/318

#RUNS 3 4 4 3 1

Analysis Germline Germline Somatic Germline Germline

Library preparation Ion Chef Ion Chef Ion Chef Ion Chef Ion Chef

Template+chip loading Ion Chef Ion Chef Ion Chef Ion Chef Ion Chef

ISP loading 82% 85% 89% 92% 91%

Total reads 3.36M 3.57M 5.28M 5.62M 5.56M

Polyclonal 32% 31% 41% 42% 54%

Usable reads 65% 67% 53% 54% 42%

Statistics per sample

Mapped Reads 410034 221423 643273 342799 170128

On Target 95.4% 95.6% 95.3% 95.3% 95.4%

Amplicons>50X 99.9% 99.9% 100% 100% 100%

Uniformity 98.4% 99.6% 99.5% 99.9% 99.9%

Mean Depth 1852 1228 2755.3 1545 763.9

29%

Tagliafico ESHG 2017.pptm 03/03/2017 Oncomine BRCA performances upon different experimental settings


#samples/Chip 8/316 16/316 8/318 16/318 32/318

#RUNS 3 4 4 3 1

Analysis Germline Germline Somatic Germline Germline

Library preparation Ion Chef Ion Chef Ion Chef Ion Chef Ion Chef

Template+chip loading Ion Chef Ion Chef Ion Chef Ion Chef Ion Chef

ISP loading 82% 85% 89% 92% 91%

Total reads 3.36M 3.57M 5.28M 5.62M 5.56M

Polyclonal 32% 31% 41% 42% 54%

Usable reads 65% 67% 53% 54% 42%

Statistics per sample

Mapped Reads 410034 221423 643273 342799 170128

On Target 95.4% 95.6% 95.3% 95.3% 95.4%

Amplicons>50X 99.9% 99.9% 100% 100% 100%

Uniformity 98.4% 99.6% 99.5% 99.9% 99.9%

Mean Depth 1852 1228 2755.3 1545 763.9

29%

Tagliafico ESHG 2017.pptm 03/03/2017 Oncomine BRCA: somatic variant detection


35.5%



38.7%



Control sample (i.e. normal germline)

Test sample (i.e. somatic)

Test sample after Noise Subtraction & Submission

FORWARD REVERSE

41.9%

Tagliafico ESHG 2017.pptm 03/03/2017 Oncomine BRCA: CNV detection


RUN Nr. of samples Nr. of CNV Concordance

with MLPA

2016_RUN42 16pz 3 100%

2017_RUN4 16pz 1 100%

2017_RUN7 8pz 2 100%

TOTAL 40 6

Data from 3 runs with 40 samples from our lab (already tested by MLPA for CNV) were analyzed in blind by Thermofisher R&D using the new Ion Reporter CNV tool on germline samples

45.2%



Representative Normal Sample: 2016 Run42 Barcode 126

48.4%



2016 Run 42 Barcode 108: BRCA1 Exon18-20 Deletion

51.6%



2016 Run 42 Barcode 108: BRCA1 Exon18-20 Deletion: MLPA

54.8%



2017 Run 7 Barcode 122: BRCA1 Exon2 Deletion

58.1%



2017 Run 7 Barcode 122: BRCA1 Exon2 Deletion: MLPA

61.3%



2017 Run 7 Barcode 123: BRCA1 Exon 4-9 Deletion

64.5%



2017 Run 7 Barcode 123: BRCA1 Exon 4-9 Deletion: MLPA

67.7%



2016 Run 42 Barcode 105: BRCA2 Exon 4-26 Duplication

71%



2016 Run 42 Barcode 105: BRCA2 Exon 4-26 Duplication: MLPA

74.2%



2016 Run 42 Barcode 105: BRCA2 Exon 4-26 Duplication: The tandem duplication description

77.4%



2016 Run 42 Barcode 105: BRCA2 Exon 4-26 Duplication: The tandem duplication description A B C D

4 kb

3 kb

80.6%

15

10

10

14

14

20

15

15

15

2

2

2

0 5 10 15 20 25 30 35 40 45 50

Oncomine™BRCAResearchAssay(2pools)(Chef+Chef)

AmpliSeqBRCA1andBRCA2CommunityPanel(3pools)(Manual+Chef)

AmpliSeqBRCA1andBRCA2CommunityPanel(3pools)(Manual+IonOneTouch)

HOURS

Librarypreparation Templateenrichment&chiploading PGMcleaning,Sequencingrun,Rawdataprocessing DataAnalysis&Variantcalling

Tagliafico ESHG 2017.pptm 03/03/2017 Processing times


AmpliSeq BRCA1 and BRCA2 Community Panel (3 pools) (Manual+IonOneTouch)

AmpliSeq BRCA1 and BRCA2 Community Panel (3 pools) (Manual+IonChef)

Oncomine™BRCA Research Assay (2 pools) (Chef+Chef)

Day 1 Day 2 Day 3 Day 4 Night 4

Day 1 Night 2 Day 3 & Night 3

Day 1 & Night 1 Night 2 Day 3 & Night 3

Times to data (16 samples/2 chips)

83.9%

1

9

9

0,75

0,75

6

3

3

3

1

1

1

0 2 4 6 8 10 12 14 16 18 20




HOURS

Librarypreparation Templateenrichment&chiploading PGMcleaning,Sequencingrun,Rawdataprocessing DataAnalysis&Variantcalling






Hands-on times (16 samples/2 chips)

87.1%

3,5

3,5

4,5

0,5

0,5

0,5

0,5

2,5

2,5

1,5

1,5

0,5

0,5

0,5

0 1 2 3 4 5 6 7 8 9 10




DAYS

NGS Interpretation Sangerforamplicons@lowcoverageandvariantvalidation MLPA Reporting






Times to report

90.3%

Tagliafico ESHG 2017.pptm 03/03/2017 Summary


OncomineTM BRCA Research Assay:

• High uniformity of coverage across all target regions (99.5%) • 100% of target regions with at least 50X at low mean depth per sample • Totally automated workflow (time saving and reproducibility) • Suitable for both germline and somatic analysis • Detection of 5% and below allele burden for somatic mutation • Detection of CNV in germline sample • Allow to report validated results within 5 days

93.5%

Tagliafico ESHG 2017.pptm 03/03/2017 Acknowledgements


Elena Tenedini Isabella Bernardis Lucia Artuso Marco Marino Laura Cortesi

Rosella Petraroli Andrea Lucchetti Chiara Magnanini Giorgio Pea Fabio Raffaldi Jon Sherlock Charles Scafe

96.8%

Tagliafico ESHG 2017.pptm 03/03/2017


Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

100%

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Implementation of BRCA Oncomine panel Enrico Tagliafico · 2020. 6. 13. · A m pliSeq BRC A 1 and...

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