evaluation of the qiaseq™ investigator snp id · evaluation of the qiaseq™ investigator snp id...
TRANSCRIPT
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
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)
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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
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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
12
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
13
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
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Peter M. Schneider
Theresa E. Gross
Institute of Legal Medicine
Cologne, Germany
Acknowledgment
Thank you for your attention.
20
Chris Phillips
Maria de la Puente
Forensic Genetics Unit
Santiago de Compostela
Spain
Ben Turner
Keith Elliot