Silicon Biosystems, SpA www.siliconbiosystems.com Silicon Biosystems International Sales Meeting Bologna, June 11 th 2012 Genetic Analysis of Individual Cells
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