Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogeneous Cellular Populations

1. Sample to Insight
230238403/2016
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
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)
WGABulk
Bulk(gDNA)
WGABulk
sc1
sc2
sc3
sc4
sc5
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)
WGABulk
Bulk(gDNA)
WGABulk
sc1
sc2
sc3
sc4
sc5
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)
WGABulk
Bulk(gDNA)
WGABulk
sc1
sc2
sc3
sc4
sc5
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