evaluation of the qiaseq™ investigator snp id · evaluation of the qiaseq™ investigator snp id...

Evaluation of the QIAseq™ Investigator SNP ID

Panel using a semiconductor-based NGS System

7th QIAGEN Investigator Forum 7-8th of March 2018 Lisbon, Portugal

Dr. Theresa E. Gross

Institute of Legal Medicine

University Hospital Cologne

Cologne, Germany

theresa.gross@uk-koeln.de

Overview

• Manual workflow – comparison

• Compatibility with semi-conductor sequencing

• Concordance with CE and MPS data

• Problematic SNPs

• Sensitivity

• Mixtures

2

MPS experience

3

Precision ID Identity Panel

preliminary version (HID v2.2)

EUROFORGEN Global

ancestry-informative SNP Panel

Ion PGM™ Workflow

4

Step 1 Step 2 Step 3

Library Preparation Template Preparation Sequencing

Data

Analysis

Step 4

QiaSeq™ Workflow

5

Step 1 Step 2 Step 3

Template Preparation Sequencing

Data

Analysis

Step 4

Biomedical Genomics

Workbench

Library Preparation

Library Preparation I

1. Library preparation Ion PGM™

a) Target enrichment

b) Primer digest

c) Adapter ligation/barcoding

d) 1st purification

e) Library amplification

f) 2nd purification

g) Library quantification

2. Template preparation

3. Sequencing

4. Data Analysis

1. Library preparation QIAseq™

a) Fragmentation

b) UMI/Adapter ligation

c) 1st cleanup

d) Target enrichment

e) 2nd cleanup

f) Universal PCR

g) 3rd cleanup

h) Library quantification

2. Template preparation

3. Sequencing

4. Data Analysis

Library Preparation II

1. Library preparation Ion PGM™

a) Target enrichment

b) Primer digest

c) Adapter ligation/barcoding

d) 1st purification

e) Library amplification

f) 2nd purification

g) Library quantification

2. Template preparation

3. Sequencing

4. Data Analysis

1. Library preparation QIAseq™

a) Fragmentation

b) UMI/Adapter ligation

c) 1st cleanup

d) Target enrichment

e) 2nd cleanup

f) Universal PCR

g) 3rd cleanup

h) Library quantification

2. Template preparation

3. Sequencing

4. Data Analysis

Library Preparation III

SNP detection with UMIs

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Compatibility

• QIAseq™ chemistry compatible with semiconductor-based NGS

• Percentage of Ion Sphere Particles (ISPs) after template

preparation within optimal range of 10-30% (11%)

• Primary data analysis (raw seq data to UBAM or fastq files)

10

Concordance I

CE data - SNPforID SNaPshot 49plex

• 48 SNPs in common with QIAseq™ SNP ID Panel

• 98% concordant genotypes

• 3 discordant genotypes in rs737681 & rs826472 due to

misinterpretation of CE data

11

Concordance II

Precision ID Identity Panel preliminary version (HID v2.2)

• 5 samples previously typed with another ID SNP MPS Panel

including 1 mixture

• 135 SNPs in common with QIAseq™ SNP ID Panel

• 97% concordant genotypes (1 sample excluded)

• 1 sample with high no-call rate (12% in comparison to an

average 2% no-calls), all due to low coverage of UMI (< 10x)

• Discordant genotypes mainly due to deletions, e.g.

rs2073383

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Concordance III

QIAseq™ SNP ID Panel analysed with HID Genotyper

• 86% concordant genotypes

• 2% no-call QIAseq™

• 1% no-call HID Genotyper

• 11% discordant genotypes

– HID Genotyper heterozygous vs. QIAseq™ homozygous

due to skewed allele read frequencies, e.g. rs727811 or

rs2399332

– 2 SNPs problematic in both analysis tools:

rs430046 & rs445251

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Problematic SNPs - IGV

14 rs727811

NA07000

12ng

IGV browser

rs727811 – Genotypes

HID Genotyper: GT

CLC Biomedical: TT

True genotype: TT

Problematic SNPs - CLC

15 rs727811

NA07000

12ng

CLC Biomedical Workbench

rs727811 – Genotypes

HID Genotyper: GT

CLC Biomedical: TT

True genotype: TT

rs430046 - IGV

16

rs430046

NA07000

12ng

rs430046 – Genotypes

HID Genotyper: CT

CLC Biomedical: T?

True genotype: TT

IGV browser

rs430046 - CLC

17

rs430046

NA07000

12ng

rs430046 – Genotypes

HID Genotyper: CT

CLC Biomedical: T?

True genotype: TT

CLC Biomedical Workbench

Sensitivity

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12 ng input

1 ng input Negative control

0.250 ng input

Dilution series of Coriell control NA07000: 1ng down to 10pg

Mixtures

• Mixture female-male ratio 2:1

• Total of 60 ng DNA input

• Average coverage 107x (11-661x)

• 98 genotypes concordant with expected mixed profile

• 37 alleles of minor contributor not detected

• No reference genotypes for 4 SNPs

• 1 SNP removed from QIAseq™ SNP ID Panel

19

Peter M. Schneider

Theresa E. Gross

Institute of Legal Medicine

Cologne, Germany

theresa.gross@uk-koeln.de

Acknowledgment

Thank you for your attention.

20

Chris Phillips

Maria de la Puente

Forensic Genetics Unit

Santiago de Compostela

Spain

Ben Turner

Keith Elliot