Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogeneous Cellular...
1
Sample to Insight 2302384 03/2016 Ryoko Shimada 1 , Kathrin Wolf 2 , Courtney Nadeau 2 , Rumeysa Akinci-Tolun 2 , Christian Grunwald 2 , Siegfried Hauch 3 and Nan Fang 2 1 QIAGEN K.K. 2 QIAGEN GmbH 3 QIAGEN Hannover Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogeneous Cellular Populations Introduction Genomic analyses at the single-cell level shed light on the genes and their functions in each individual cell and give valuable information on the fundamental mechanisms of cellular function. Recent developments in single cell sequencing technologies have enabled single-cell genomic analysis at single nucleotide resolution and revealed the cellular heterogeneity among presumably homogeneous cell populations. Single-cell sequencing has become a powerful tool in cancer research (for early detection and monitoring of rare non-identical circulating tumor cells), in immunology, stem cell research, and preimplantation genetic diagnosis. Targeted sequencing, the selective enrichment and sequencing of a panel of genes of interest, is a sensitive and cost-effective method to analyze functionally relevant genes. The targeted sequencing at single-cell level is especially useful in cancer research, due to the high heterogeneity of the tumor cells and relatively small numbers of significantly mutated genes (SMGs) in all cancer types. This poster describes an optimized workflow for streamlined targeted sequencing at the single-cell level or with enriched circulating tumor cells (CTCs) that can be used for liquid biopsy. The targeted single cell sequencing workflow combines reliable, multiple displacement amplification (MDA)-based single-cell whole genome amplification (WGA), multiplex PCR-based targeted enrichment, and a fast one-step sequencing library construction protocol. We demonstrated that this method can be used to sensitively and reliably detect oncogene mutations in a heterogeneous cellular population. Good Sequencing Metrics To further evaluation of the library quality, we analyzed specificity and uniformity of bulk and single cell samples: Outstanding specificity uniformity and coverage were achieved on sequencing results for all samples. Samples were either DNA from individual cells or DNA from bulk cells that underwent WGA, or un-amplified bulk genomic DNA. Conclusions • Efficient targeted sequencing workflow • Reliable single cell amplification, specific and uniform target enrichment, and innovative 1-step amplicon library prep • Sensitive mutation detection for either single cells or mixed cell populations • Powerful liquid biopsy tool for cancer mutation profiling • Isolated single CTCs • Enriched CTCs Trademarks: QIAGEN ® , QIAamp ® , REPLI-g ® , GeneRead ™ (QIAGEN Group); CellRaft ™ (Cell Microsystems, Inc.); MiSeq ® , NextSeq ® (Illumina, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2016 QIAGEN, all rights reserved. Detection of Variant in Single Cells The figure shows the mutation rates for various DNA samples detected by the GeneRead Targeted Panels V2 (Human Clinically Relevant Tumor Panel). Previously known variants in LoVo cell (KRAS G13D) and HT29 cell (BRAF V600E) were detected by next-generation sequencing (NGS). It was possible to determine mutations even in samples with only 5% of mutated gDNA, showing high method sensitivity. Mutation rate of single cells, bulk DNA and mixtures of bulk DNA. Mutation rate % Mutation rate % Bulk (gDNA) WGA Bulk Bulk (gDNA) WGA Bulk sc 1 sc 2 sc 3 sc 4 sc 5 5/95 20/80 50/50 80/20 95/5 80 60 40 20 0 LoVo HT-29 isolated single cells Samples LoVo/HT-29 Bulk mix KRAS p.G13D BRAF p.V600E Convenient Library Preparation Method The new QIAseq 1-Step Amplicon Library Kit (cat. no. 180412) is designed specifically for constructing sequencing libraries with multiplex PCR products. It is 30 minute single-tube room-temperature library prep for targeted resequencing and amplicon sequencing. A The BioAnalyzer trace shows the expected size shift (+) for illumina libraries after library preparation and amplification. Neither non-specific product nor adapter dimer were detected. A : Bioanalyzer trace of GeneRead Panel PCR Product. B : Bioanalyzer trace of sequencing library prepared with new QIAseq 1-Step Amplicon Library Kit. 120 min , 3 reagent addition steps, 3 Different Temperatures 30 min, one room temperature step A-Addition 37°C HiFi Library Amplification End Repair 25°C Size Selection QIAseq 1-Step Amplicon Library Prep Multiplex PCR Products Multiplex PCR Products Adapter Ligation 25°C HiFi Library Amplification Size Selection 1-Step Library Prep 75°C Heat Inactivation 75°C Heat Inactivation GeneRead DNA Library Prep B Material and Methods We designed two model of experimental workflow to detect cancer mutations via liquid biopsy from blood: 1. Isolated single cell Single cells were isolated from a mixture of two different colorectal cancer cell line (HT- 29 and Lovo) using CellRaft ™ Array (Cell Microsystems). Released rafts carrying individual cells were transferred to PCR tubes and single cell genome DNA was amplified with a MDA-based approach using REPLI-g ® Single Cell Kit (QIAGEN). 2. Enriched CTC mimic cells For proof-of-principle experiment we controlled the percentage of the tumor cells in the blood cell background by spike breast cancer cell line (MCF 7) into blood cells. We treated this sample as a mimic of the enriched CTC. * GeneRead DNAseq Targeted Panel (QIAGEN) which is a multiplex PCR target enrichment panel was used to amplify exonic regions of cancer relevant genes. Isolated single cell Enriched CTC mimic cells Single Cell Isolation Isolated single cell (HT-29 and Lovo) by Cell Raft System Adnagen mimic Sample Prep Spiked MCF 7 Cells into 1000 Blood Cells at 5% NGS run MiSeq ® NGS run MiSeq/NextSeq ® One-step library prep Library for NGS was constructed by QIAseq 1-Step Amplicon Library Kit Web based data analysis Data was analyzed by GeneRead DNAseq variant calling service on QIAGEN website Whole genome amplification Single cell genome was amplified with REPLI-g Single Cell Kit DNA Purification QIAamp ® gDNA Prep Target enrichment by multiplex gene panel PCR GeneRead DNAseq Targeted Panels V2* (Human Clinically Relevant Tumor Panel) Target enrichment by multiplex gene panel PCR GeneRead DNAseq Targeted Panels V2* (Human Breast Cancer Panel) 1 2 Enriched CTC mimic cells We did the the proof-of-principle experiment for targeted sequencing of the CTCs enriched with AdnaGen (QIAGEN Hannover). The AdnaGen CTC technology can enrich tumor cell using magnetic particles conjugated with an antibody mixture to ensure CTC capturing even in case of EpCAM loss. We could successfully detect 5% previously known variants in MCF 7 Cells as the CTC mimic by GeneRead ™ DNAseq Targeted Panels V2 (Human Breast Cancer Panel) in combination with the QIAseq 1Step Amplicon Library Kit with either moderate or high sequencing depth (1391X median depth on MiSeq or 8518X median depth on NextSeq). Mutation rate of spiked MCF 7 Cells into 1000 Blood Cells at 5%. The names of the representative genes and mutations–known for MCF7 cells–are listed under the X-axis. (MUC16 and RET) Tumor Mutation Detection Allele Frequency (%) 8 7 6 5 4 3 2 1 0 Theoretical % % detected with NextSeq Run % detected with miSeq Run MUC16 (P12220L) RET (R189H) A : Specificity is calculated as percentage of reads mapped to target region of interest out of total number of reads per run. B : Coverage uniformity is measured as percentage of bases in the region of interest covered at least 0.1x or 0.2x mean coverage depth. Specificity Reads% aligned to target region Bulk (gDNA) WGA Bulk Bulk (gDNA) WGA Bulk sc 1 sc 2 sc 3 sc 4 sc 5 5/95 20/80 50/50 80/20 95/5 100 80 60 40 20 0 LoVo HT-29 isolated single cells Samples LoVo/HT-29 Bulk mix Uniformity Base% covered Bulk (gDNA) WGA Bulk Bulk (gDNA) WGA Bulk sc 1 sc 2 sc 3 sc 4 sc 5 5/95 20/80 50/50 80/20 95/5 100 80 60 40 20 0 LoVo HT-29 isolated single cells Samples LoVo/HT-29 Bulk mix 0.1x mean 0.2x mean A B 2302384_SPOS_ICHG_SingleCell.indd 1 2016/03/31 18:02
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Transcript of Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogeneous Cellular...
Sample to Insight
2302
384
03
/201
6
Ryoko Shimada1, Kathrin Wolf2, Courtney Nadeau2, Rumeysa Akinci-Tolun2, Christian Grunwald2, Siegfried Hauch3 and Nan Fang2
1 QIAGEN K.K. 2 QIAGEN GmbH 3 QIAGEN Hannover
Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogeneous Cellular Populations
IntroductionGenomic analyses at the single-cell level shed light on the genes and their functions in each individual cell and give valuable information on the fundamental mechanisms of cellular function.
Recent developments in single cell sequencing technologies have enabled single-cell genomic analysis at single nucleotide resolution and revealed the cellular heterogeneity among presumably homogeneous cell populations. Single-cell sequencing has become a powerful tool in cancer research (for early detection and monitoring of rare non-identical circulating tumor cells), in immunology, stem cell research, and preimplantation genetic diagnosis.
Targeted sequencing, the selective enrichment and sequencing of a panel of genes of interest, is a sensitive and cost-effective method to analyze functionally relevant genes. The targeted sequencing at single-cell level is especially useful in cancer research, due to the high heterogeneity of the tumor cells and relatively small numbers of significantly mutated genes (SMGs) in all cancer types.
This poster describes an optimized workflow for streamlined targeted sequencing at the single-cell level or with enriched circulating tumor cells (CTCs) that can be used for liquid biopsy. The targeted single cell sequencing workflow combines reliable, multiple displacement amplification (MDA)-based single-cell whole genome amplification (WGA), multiplex PCR-based targeted enrichment, and a fast one-step sequencing library construction protocol. We demonstrated that this method can be used to sensitively and reliably detect oncogene mutations in a heterogeneous cellular population.
Good Sequencing MetricsTo further evaluation of the library quality, we analyzed specificity and uniformity of bulk and single cell samples: Outstanding specificity uniformity and coverage were achieved on sequencing results for all samples. Samples were either DNA from individual cells or DNA from bulk cells that underwent WGA, or un-amplified bulk genomic DNA.
Conclusions• Efficient targeted sequencing workflow
• Reliable single cell amplification, specific and uniform target enrichment, and innovative 1-step amplicon library prep
• Sensitive mutation detection for either single cells or mixed cell populations
• Powerful liquid biopsy tool for cancer mutation profiling
• Isolated single CTCs
• Enriched CTCs
Trademarks: QIAGEN®, QIAamp®, REPLI-g®, GeneRead ™ (QIAGEN Group); CellRaft ™ (Cell Microsystems, Inc.); MiSeq®, NextSeq® (Illumina, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.© 2016 QIAGEN, all rights reserved.
Detection of Variant in Single CellsThe figure shows the mutation rates for various DNA samples detected by the GeneRead Targeted Panels V2 (Human Clinically Relevant Tumor Panel). Previously known variants in LoVo cell (KRAS G13D) and HT29 cell (BRAF V600E) were detected by next-generation sequencing (NGS). It was possible to determine mutations even in samples with only 5% of mutated gDNA, showing high method sensitivity.
Mutation rate of single cells, bulk DNA and mixtures of bulk DNA.
Mutation rate %Mutation rate %
Bulk
(gD
NA
)
WG
A B
ulk
Bulk
(gD
NA
)
WG
A B
ulk
sc 1
sc 2
sc 3
sc 4
sc 5
5/95
20/8
0
50/5
0
80/2
0
95/5
80
60
40
20
0
LoVo HT-29 isolated single cells
Samples
LoVo/HT-29 Bulk mix
KRAS p.G13DBRAF p.V600E
Convenient Library Preparation MethodThe new QIAseq 1-Step Amplicon Library Kit (cat. no. 180412) is designed specifically for constructing sequencing libraries with multiplex PCR products. It is 30 minute single-tube room-temperature library prep for targeted resequencing and amplicon sequencing.
AThe BioAnalyzer trace shows the expected size shift (+) for illumina libraries after library preparation and amplification. Neither non-specific product nor adapter dimer were detected.A : Bioanalyzer trace of GeneRead Panel PCR Product. B : Bioanalyzer trace of sequencing library prepared with new QIAseq 1-Step Amplicon Library Kit.
120 min , 3 reagent addition steps, 3 Different Temperatures
30 min, one room temperature step
A-Addition37°C
HiFi Library Ampli�cationEnd Repair
25°C
Size Selection
QIAseq 1-Step Amplicon Library Prep
Multiplex PCR Products
Multiplex PCR Products
Adapter Ligation25°C
HiFi Library Ampli�cation
Size Selection
1-Step Library Prep
75°C HeatInactivation
75°C HeatInactivation
GeneRead DNA Library Prep
B
Material and MethodsWe designed two model of experimental workflow to detect cancer mutations via liquid biopsy from blood:
1. Isolated single cell Single cells were isolated from a mixture of
two different colorectal cancer cell line (HT-29 and Lovo) using CellRaft ™ Array (Cell Microsystems). Released rafts carrying individual cells were transferred to PCR tubes and single cell genome DNA was amplified with a MDA-based approach using REPLI-g® Single Cell Kit (QIAGEN).
2. Enriched CTC mimic cells For proof-of-principle experiment we
controlled the percentage of the tumor cells in the blood cell background by spike breast cancer cell line (MCF 7) into blood cells. We treated this sample as a mimic of the enriched CTC.
* GeneRead DNAseq Targeted Panel (QIAGEN) which is a multiplex PCR target enrichment panel was used to amplify exonic regions of cancer relevant genes.
Isolated single cell Enriched CTC mimic cells
Single Cell Isolation
Isolated single cell (HT-29 and Lovo) by Cell Raft System
Adnagen mimic Sample Prep
Spiked MCF 7 Cells into 1000 Blood Cells at 5%
NGS run
MiSeq®
NGS run
MiSeq/NextSeq®
One-step library prep
Library for NGS was constructed by QIAseq 1-Step Amplicon Library Kit
Web based data analysis
Data was analyzed by GeneRead DNAseq variant calling service on QIAGEN website
Whole genome amplification
Single cell genome was amplified with
REPLI-g Single Cell Kit
DNA Purification
QIAamp® gDNA Prep
Target enrichment by multiplex gene panel PCR
GeneRead DNAseq Targeted Panels V2* (Human Clinically
Relevant Tumor Panel)
Target enrichment by multiplex gene panel PCR
GeneRead DNAseq Targeted Panels V2* (Human Breast
Cancer Panel)
1 2
Enriched CTC mimic cellsWe did the the proof-of-principle experiment for targeted sequencing of the CTCs enriched with AdnaGen (QIAGEN Hannover). The AdnaGen CTC technology can enrich tumor cell using magnetic particles conjugated with an antibody mixture to ensure CTC capturing even in case of EpCAM loss. We could successfully detect 5% previously known variants in MCF 7 Cells as the CTC mimic by GeneRead ™ DNAseq Targeted Panels V2 (Human Breast Cancer Panel) in combination with the QIAseq 1Step Amplicon Library Kit with either moderate or high sequencing depth (1391X median depth on MiSeq or 8518X median depth on NextSeq).
Mutation rate of spiked MCF 7 Cells into 1000 Blood Cells at 5%.The names of the representative genes and mutations–known for MCF7 cells–are listed under the X-axis. (MUC16 and RET)
Tumor Mutation DetectionAllele Frequency (%)
8
7
6
5
4
3
2
1
0
Theoretical %% detected with NextSeq Run
% detected with miSeq Run
MUC16 (P12220L) RET (R189H)
A : Specificity is calculated as percentage of reads mapped to target region of interest out of total number of reads per run. B : Coverage uniformity is measured as percentage of bases in the region of interest covered at least 0.1x or 0.2x mean coverage depth.
Speci�cityReads% aligned to target region
Bulk
(gD
NA
)
WG
A B
ulk
Bulk
(gD
NA
)
WG
A B
ulk
sc 1
sc 2
sc 3
sc 4
sc 5
5/95
20/8
0
50/5
0
80/2
0
95/5
100
80
60
40
20
0
LoVo HT-29 isolated single cells
Samples
LoVo/HT-29 Bulk mix
UniformityBase% covered
Bulk
(gD
NA
)
WG
A B
ulk
Bulk
(gD
NA
)
WG
A B
ulk
sc 1
sc 2
sc 3
sc 4
sc 5
5/95
20/8
0
50/5
0
80/2
0
95/5
100
80
60
40
20
0
LoVo HT-29 isolated single cells
Samples
LoVo/HT-29 Bulk mix
0.1x mean0.2x mean
A
B
2302384_SPOS_ICHG_SingleCell.indd 1 2016/03/31 18:02
