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