Download Balanced, robust whole genome amplification from single cells

Balanced, robust whole genome
amplification from single cells
Ampli1™ WGA Kit balanced allelic
amplification
Every Cell Has A Story To Tell™
The Ampli1™ Whole Genome Amplification (WGA)
Kit has been developed and optimized to deliver a
balanced and complete amplification of the total DNA
content of a single cell. 1
When working with pure, single cells, achieving
balanced amplification of both alleles at every single
locus is very challenging.
On a whole genome sequencing test 2, The Ampli1™
WGA Kit has demonstrated a significantly lower ADO
(allelic drop out) rate (2%) as compared with Multiple
Displacement Amplification (MDA) (up to 65%).
In addition, the percentage of genomic sequence
amplified by the Ampli1™ WGA method is two-fold
higher as compared with other PCR based methods
(74% vs. 36%). 2
Fig. 1
Single Cell
Other methods
Ampli1™ WGA Kit
Site specific digestion
• ROBUST:
Works on fresh and fixed single cells
No site specific digestion
NNNNTTAANNNN
NNNNAATTNNNN
Random priming
Allele
Allele
Digested
fragments
Uncontrolled amplification
Single primer amplification
Results
Results
Unbalanced allelic amplification,
allelic drop out
Balanced allelic amplification,
low allelic drop out
Cell 1
Cell 2
Cell 3
Cell 4
Balanced allele amplification
of different single cells
Cell n
Cell 1
Cell 2
Cell 3
Cell 4
• RELIABLE:
Single primer-mediated PCR ensures balanced
amplification
• REPRODUCIBLE:
Single tube, no-precipitation protocol
minimizes template loss
Allele
Most WGA methods use random priming
and uncontrolled amplification, causing frequent
allelic dropout due to preferential (unbalanced)
amplification of one of the two alleles.
As a result, such WGA methods are only reliable
for genetic analysis of variants (SNP) when
working with >100 pooled cells or for copy number
variation analysis (CNV) when working with >10 cells.
Instead, the DNA library generated by the Ampli1™
WGA method is ideally suited for downstream
analysis of both SNPs and CNV even from single cells,
due to its low rate of preferential amplification,
as shown in Fig. 1.
Key features of the Ampli1™ WGA Kit
Cell n
Unbalanced allele amplification
of different single cells
The illustration shows how balanced amplification is achieved with the Ampli1™ WGA
method compared to other methods in which preferential amplification of one allele
over another can result in allelic drop out (ADO). With the Ampli1™ WGA Kit,
following cell lysis, DNA is digested with a restriction enzyme and adaptors are
ligated onto the DNA fragments. Amplification, performed in the same tube,
is mediated by a single highly specific PCR primer for all 19 million fragments.
Reliable sequencing of DNA from single cells after amplification
with Ampli1™ WGA Kit 8
DNA may be derived from fresh, fixed and/or
stained cells, including FFPE derived tumor
and stromal cells. 3,4,5,6
Additionally, hundreds of positive experiments
have been executed on single cells isolated from
multiple kinds of samples: WBCs, CTCs, sperm
cells, CSF, stem cells, neural cells and fetal cells.
Furthermore, results obtained with the Ampli1™
WGA Kit did not reveal any significant decrease
of WGA quality on cells fixed and/or stored for
a long time. 7
Mean SCR % on Single Cells
Ampli1™ WGA Kit is ideal for Various Cell Types
100%
Robust amplification
from fixed or live cells
90%
80%
STR Call Rates (SCR) from
multiplexed analysis of 11
STR loci show the Ampli1™
WGA Kit can be equally
used for DNA amplification
of single live or fixed cells.
70%
60%
50%
40%
30%
20%
Single tumor and white blood cells (WBCs) were isolated from controls spiked with tumor cells and cancer patient
samples using Menarini Silicon Biosystems’ DEPArray™ system. Genomic DNA from each individual tumor cell and WBC
were amplified using the Ampli1™ WGA Kit and then sequenced with the Ion Torrent® AmpliSeq™ Custom Panel.
The sequence table below shows the detection of homozygous (HM) and heterozygous (HT) gene mutations.
The results demonstrate very low allelic drop out (ADO), no false positive calls, and a balanced detection of variant
and wild type alleles in both fixed and fresh cells.
The expected heterozygous PIK3CA mutation was found in all MCF7 replicates. SW480 resulted in several homozygous
mutations, including the expected KRAS, TP53 and KIT mutations.
10%
0%
Mean SCR %
Live
2% PFA
Streck Cell
PreservativeTM
97%
75%
77%
6%
11%
11%
St. Dev.
gDNA
Ampli1™ WGA Kit One-Day Protocol
Following cell lysis, DNA is digested with
a restriction enzyme and adaptors are ligated
onto the DNA fragments.
Amplification, performed in the same tube,
is mediated by a single highly specific
PCR primer for all fragments.
STEP 1
STEP 2A
Cell Lysis:
DNA Digestion:
Add Lysis
Reaction
Mix to each
sample
and incubate
Hands-on Time
Total Time
10’
Add Digestion
Reaction Mix to
the same tube
and incubate
10’
1h 40’
20’
15’
1h 15’
STEP 2B
Preannealing:
Prepare Preannealing Reaction Mix and incubate
STEP 3
Adaptor
Ligation:
Add ligation
Reaction mix
and incubate
10’
STEP 4
Amplification:
Add Primary
PCR Reaction
Mix and amplify
10’
1h 10’
40’
3h 30’
6h 40’
fixed
fixed
fixed
fixed
fixed
fixed
fixed
live
live
live
fixed
fixed
fixed
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
WGA
Gene
COSMIC ID
Allele Call
MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7 SW480 SW480 SW480 SW480 SW480 SW480
KRAS
COSM520
Homozygous
100
100
100
100
100
100
TP53
COSM10660
Homozygous
100
100
100
100
100
100
SMAD4
COSM14167
Homozygous
100
100
100
100
100
PIK3CA
COSM763
Heterozygous
PDGFRA
COSM22413
Homozygous
100
100
100
100
100
100
KIT
COSM28026
Homozygous
100
100
100
100
100
100
51
55
65
44
58
71
66
59
Spiked-in single MCF-7 and SW480 tumor cells were recovered, amplified and then sequenced.
The table above shows a portion of the results from interrogation of ~2800 COSMIC hotspot mutations in 54 genes from a modified version of Ion AmpliSeq™
Cancer Hotspot Panel V2, adapted for use after Ampli1™ WGA Kit. 8
Maximum Flexibility for Downstream
Analyses
Amplified DNA generated with the Ampli1TM WGA Kit
can be used for the most demanding laboratory
workflow and genomic applications and is suitable
with the following methods and analysis:
Product Information Description
Ampli1™ QC Kit (Genome Integrity Index)
Description
The Ampli1TM QC Kit provides a useful quality control check of the WGA procedure by utilizing a PCR-based assay
to establish DNA integrity. By testing for the presence of one short and three long DNA fragments in the WGA library,
the quality of the DNA as starting material for downstream analyses can be assured. Cells with good quality DNA
typically produce 3 or 4 PCR bands, while fixation of cells or cells with degraded DNA will show fewer QC bands.
It has been proved that the number of the bands revealed is useful to define a “Genome Integrity Index” for predicting
success of various downstream analytical methods and for assessing the quality of the starting DNA. 9
• Ampli1TM WGA Kit and Ampli1TM QC Kit
are available in 50 rxn and 200 rxn respectively.
Methods
• Sanger Sequencing
• aCGH
• qPCR
PCR Product Identification
Ampli1™ Sequencing Kits
• Next Generation Sequencing
Analysis
• Sequencing
• SNP and Mutation Detection
• STR
• CNV
• Loss of Heterozygosity
Menarini Silicon Biosystems has developed Sanger
Sequencing kits for the detection of most common
cancer-related mutations, making possible their
charaterization in single cells.
The Ampli1TM Sequencing Kits are designed in order
to be fully compatible with the DNA libraries generated
with Ampli1TM WGA products, and take advantage
of its specific features of being generated with
a restriction enzyme.
The Ampli1TM Sequencing Kits are PCR-based assays
designed to be highly sensitive and specific.
Along with the Ampli1TM WGA Kit, the Ampli1TM
Sequencing Kits, represent a true, accurate option
available for the detection of the mutation in single cells.
• Ampli1TM PIK3CA Seq Kit 50rxn
• Ampli1TM EGFR Seq Kit 20rxn
• Ampli1TM KRAS Seq Kit 100rxn
• Ampli1TM BRAF Seq Kit 100rxn
• Ampli1TM ALK Seq Kit 25rxn
Example of agarose gel electrophoresis of Ampli1™ QC Kit products
Target
Chromosome
Amplicon Length (bp)
A
12p
91
B
5p
108-166
C
17p
299
D
6p
614
PCR expected product Length
References from content:
1 Klein CA, et. al., Proc. Natl. Acad. Sci.1999 - “Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells”
2 Binder V, et. al., Human Mutation 2014 - “A new workflow for whole genome sequencing of single human cells”
3 Carpenter E, et. al., Frontiers in Oncology 2014 - “Dielectrophoretic capture and genetic analisys of single neuroblastoma tumor cells”
4 Fernandez SV, et. al., Breast Cancer Research 2014 - “TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast
cancer patients”
5 Pestrin M, et. al., Molecular Oncology 2014 - “Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast
cancer patients”
6 Hodgkinson CL, et. al., Nature Medicine 2014 - “Tumorigenicity and genetic profiling of Circulating Tumor Cells in Small Cell Lung Cancer”
7 Neves RPL, et. al., Clinical Chemistry 2014 - “Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating
Tumor Cells from Patients with Metastatic Breast Cancer”
8 Buson G, et. al., Poster ACTC Crete 2014 - “Simultaneous, targeted copy number variation (CNV) and single nucleotide variant (SNV) detection on single CTCs
with Ampli1™ Whole Genome Amplification and Ion Torrent® AmpliSeq™ Custom Panel”
9 Polzer B, et. al., EMBO Molecular Medicine 2014 - “Molecular profiling of single circulating tumor cells with diagnostic intention”
Notices and Disclaimers: Ampli1™ WGA Kits are for Research Use Only and not intended for use in diagnostic procedures. Ampli1™ WGA Kits are not for resale
except by authorized distributors. The Ampli1™ WGA Kit is protected by U.S. and International patents. Please contact Menarini Silicon Biosystems for licensing
or other commercial terms. DEPArray™, and Ampli1™ are trademarks of Menarini Silicon Biosystems, S.p.A. Ion Torrent® and Ion AmpliSeq™ are trademarks of Life
Technologies, Inc. Streck Cell Preservatives™ is a trademark of Streck Innovations.
BR_WGA_v1.0_05/2016
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