User's Manual

In the first step of enrichment, the sample is diluted and partitioned into millions of double emulsion droplets

using the Xdrop™ instrument and the advanced microfluidics dPCR cartridge. These droplets are highly stable

and are suitable for standard PCR cycling, flow cytometry analysis and sorting. Droplets containing the target

DNA molecules are identified by a 120-160 bp targeted droplet PCR (dPCR) specific to a sequence (ID

sequence) within or adjacent to the region of interest. Positive droplets are identified by their fluorescence

and physically separated from negative droplets using a standard cell sorter. The result is an enrichment of

long single molecules comprising kilobases of DNA information.

For downstream DNA amplification of the single molecules, Samplix has developed a proprietary technology.

Each long fragment derived from the enrichment is partitioned into thousands of

single emulsion droplets

for high fidelity multiple displacement amplification in droplets (dMDA).

The Xdrop™ enrichment and amplification technology are compatible with downstream molecular biology

techniques such as short- and long-read sequencing.

Workflow overview

Droplet PCR (dPCR)

Chapter

~ 20 min Prepare dPCR mix and cartridge set up

~ 40 min dPCR droplet generation in the Xdrop™ instrument

~ 2 h dPCR in thermal cycler

Good pause point: store dPCR droplets after dPCR at 4⁰C for up to 24 h

Chapter 4

Flow cytometry

~ 15 min Sample staining and setup

~ 30 min/sample Sorting (time depends on instrument)

Proceed immediately to dMDA

Droplet Multiple Displacement Amplification (dMDA)

~ 10 min dPCR droplets break

Chapter 5

~ 20 min Prepare dMDA mix and cartridge set up

~ 45 sec dMDA droplet generation in the Xdrop™ instrument

~ 16 h (overnight) dMDA in thermal cycler

~ 10 min dMDA droplets break

Verify enrichment and proceed to library preparation and sequencing