Post on 10-Jan-2022
Silicon Biosystems, SpA
www.siliconbiosystems.com
Silicon Biosystems International Sales Meeting
Bologna, June 11th 2012
Genetic Analysis of Individual Cells
p. 2
Enabling the Downstream Analysis
Ampli1™: Whole Genome and Transcriptome
Amplification from single cells
Thorough information
Scalable resolution
– Massively parallel analysis platforms
– Targeted tests
Ampli1™ WGA Launched
Ampli1™ WTA 4Q2012
Ampli1™ Whole Genome DNA Amplification
p. 3
Enzymatic digestion
preannealing of adaptors
ligation XXXAATTXX XXXTTAAXX
Removal of unligated small primers
filling reaction at 3´
primary PCR-amplification (WGA)
proteinase K digest of single cell; inactivation of proteinase K
Source: C. Klein – University of Regensburg
Different Features of WGA Methods
WGA Kit Company Method Single
Cells
Fixed
Cells
Input
DNA
[ng]
STR WTA QC
Kit
Ampli1™ Silicon
Biosystems
LM-
PCR + + 0,01 + + +
REPLI-g™ GE MDA +/- - 10 + - -
GenomiPhi™ Qiagen MDA +/- - 10 + - -
PicoPlex Sigma-
Aldrich
IL-
UPPCR + -
0,01
- - -
p. 4
The combinations of these features represent the Ampli1™ WGA Unique Selling Point.
Ampli1™: Enabling Whole Genome and
Transcriptome Analysis from Single-Cells
Ampli1™ WGA Unique Features
Comprehensive and homogenous amplification of the
whole genome isolated from a single cell even if fixed
Robust and reproducible
Complete representation of the genome with fragments
of 0.2-2 kb
Sequence complexities of multiple primer binding sites
are avoided resulting in optimal amplification conditions
for all adapter-ligated sequences used
Possibility to combine the WGA analysis with the WTA
analysis starting from the same single cell.
p. 6
DOs and DON’Ts of Ampli1™ WGA
DOs: Start from fresh or fixed SINGLE
cells
Detect Mutation by:
– Target PCR + Sanger Sequencing
– Allelic Discimination Real Time PCR
– Next Generation Sequencing
STR Genotyping
Loss of heterozigosity (LOH) assays
Metaphase CGH & Array CGH
Whole Trascriptome Amplification
from the same Single Cell
Re-amplification of the WGA product
…
DON’Ts Design Target PCR
including possible digestion
sequences
BAC array
Future Developments
SNParray and ChIP-Seq (?)
…
p. 7
p. 8
Ampli1™ QC Kit
Multiplex Assay to Predict
Successful metaphase CGH
9
Multiplex PCR assay: three markers are selected for their accuracy to predict a successful mCGH
experiment with Ampli1™ WGA samples.
The statistical values calculated include:
– Sensitivity = true positives/ (true positives + false negatives)
– Specificity = true negatives / (true negatives+ false positives)
– Positive Predictive Value = true positives /(true positives + false positives)
– Negative Predictive Value = true negatives /(true negatives + false negatives)
Why Running the QC Kit?
To confirm the Ampli1™ WGA reaction success
for each cell
To identify the best cells in terms of homogenouse
representation of the input genome and select
them for a deeper analysis (e.g. NGS, aCGH,
mCGH)
p. 10
Silicon Biosystems, SpA
www.siliconbiosystems.com
Ampli™: Ancillary Kits for Single Cells
Downstream Analysis
Ampli1™ Product is a Library which
Contains all the Knowledge of the Cell
12
Where is my book?
Ampli1™ Single Cell Molecular Analysis Pipeline
Product Name Design Testing Manufacturing Market
Ampli1™ WGA
Ampli1™ QC kit
Ampli1™ WTA
Ampli1™ KRAS Seq
Ampli1™ BRAF Seq
Ampli1™ AR Seq
Ampli1™ PI3K Seq
Ampli1™ ALK Seq
Ampli1™ EGFR Seq
Ampli1™ Her2 CNV
Ampli1™ EGFR CNV
Ampli1™ AR CNV