Samplix XDROP-8 User Manual

Xdrop™ manual

User manual v. 2.0 released 14 Feb. 2020

Table of Contents

Chapter 1: XdropTM at a Glance page 3

Targeted enrichment overview

Workflow overview
Xdrop™ instrument overview
Required items for Xdrop™
Suggested Samplix products

Equipment and reagents for Xdrop™ supplied by the user

Chapter 2: Droplet PCR Reaction Design for Target Selection page 12

Target sequence
Primer design guidelines
Primer Testing

Chapter 3: Droplet PCR (dPCR) page 16

Preparations for dPCR
Setup of dPCR reaction
Optional: Positive control dPCR reaction
Prepare dPCR cartridge
Collect generated droplets

Chapter 4: Single DNA Molecule Detection and Sorting of Droplets page 26

Requirements for cell sorter
Notes to operator
Preparation of droplets for flow cytometry
Flow cytometry analysis and sorting of dPCR droplets
Optional: Set up flow cytometry with Cell sorter control droplets

Chapter 5: Multiple Displacement Amplification in droplets (dMDA) page 31

Break sorted droplets (if applicable)
Set up dMDA reaction
Load the dMDA cartridge
Generate dMDA droplets on the Xdrop™ instrument

Chapter 6: Evaluation of Amplification and Target Enrichment page 42

Quantify total DNA
Evaluate the enrichment of target DNA
Calculate fold enrichment of target DNA

Samplix ApS
Mileparken 28
DK – 2730 Herlev

http://www.samplix.com

Mail: [e-mail]

For research use only, not for use in diagnostic procedures. Copyright 2020. Reproduction in any form, either print or electronic,
is prohibited without written permission of Samplix ApS.

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Chapter 1: Xdrop™ at a Glance

Targeted enrichment overview

Congratulations with your new Xdrop™ instrument, which we expect will facilitate groundbreaking research.

The Xdrop™ introduces a new approach for genomic analysis. Our innovative technology enables targeted
enrichment of genomic regions in droplets. The Xdrop™ system offers sensitive and unbiased PCR-free
sample enrichment and isothermal amplification prior to downstream analysis e.g. next generation
sequencing.

Using the Xdrop™ instrument, cartridges, and reagents, DNA samples are partitioned into millions of picolitre
size droplets. The workflow includes two steps:

1. Target enrichment in double emulsion droplets (Chapter 3, Droplet PCR – dPCR)

2. Isothermal amplification in single emulsion droplets (Chapter 5, Multiple Displacement Amplification
in droplets – dMDA)

Fig. 1.1. Xdrop™ workflow including dPCR (droplet PCR) and dMDA (Multiple Displacement Amplification in

droplets).

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Chapter

Chapter 4

Chapter 5

Verify enrichment and proceed to library preparation and sequencing

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
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

single emulsion droplets

Droplet PCR (dPCR)

~ 20 min Prepare dPCR mix and cartridge set up

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~ 40 min dPCR droplet generation in the Xdrop™ instrument

~ 2 h dPCR in thermal cycler

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

~ 20 min Prepare dMDA mix and cartridge set up

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

~ 45 sec dMDA droplet generation in the Xdrop™ instrument

~ 16 h (overnight) dMDA in thermal cycler

~ 10 min dMDA droplets break

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Start button

Xdrop™ instrument overview

The Xdrop™ droplet generator instrument is compatible with Samplix dPCR cartridges for production of
double emulsion dPCR droplets and Samplix dMDA cartridges for the generation of single emulsion droplets
for amplification of DNA. When using dMDA cartridges, always use the accompanying holder. The Xdrop™
droplet generator is used for generating both dPCR and dMDA droplets and is composed
parts (see figure below):

-

A start button on the front of the instrument.

-

Touch screen: provides the means to control the droplet generator with gloved or un-gloved
hands.

-

Status LED: purple when initializing and shutting down, green when in standby, blue when
operating and yellow-green when opening and closing the drawer.

of the following

-

Drawer: holds the dPCR or dMDA cartridge.

-

USB port on the back of the instrument: connects to a USB flash drive for updating
instrument firmware.

-

Air vents on the back of the instrument: for ventilation.

-

A hardware switch on the back of the instrument.

Touch screen

Status LED

5

Specifications

Width: 25 cm / 9,8 inches
Height: 25 cm / 9,8 inches
Length: 48 cm / 18,9 inches
Weight 17 kg / 37,5 lbs.
Voltage requirements: 110 V-240 V (Line frequency; 50 - 60 Hz, Max current: 2,3 A)

Support

To find technical support, contact the support team at [e-mail]

Warranty

The Xdrop™ instrument and associated accessories are covered by a standard Samplix ApS warranty.
Contact your local Samplix ApS office for the details of the warranty.

Safety

We strongly recommend that you follow the safety specifications listed in this section and throughout this
manual.

Xdrop™ is produced to comply with Safety Requirements for Electrical Equipment for Measurement, Control,
and Laboratory Use (UL 61010-1) and complies with EU (CISPR 11, class A, group 1, 150 kHz – 30 MHz) EMC.

This equipment has also been tested and found to comply with the limits for a Class A digital device, pursuant
to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful
interference when the equipment is operated in a commercial environment. This equipment generates, uses,
and can radiate radio frequency energy and, if not installed and used in accordance with the instruction
manual, may cause harmful interference to radio communications. Operation of this equipment in a
residential area is likely to cause harmful interference in which case the user will be required to correct the
interference at his own expense.

FCC Caution: Any changes or modifications not expressly approved by the party responsible for compliance
could void the user's authority to operate this equipment.

Environment and Power Requirements

Xdrop™ operating conditions are at temperatures 20-25°C, with a relative humidity (RH) 0-75%. Xdrop™
requires a stable power supply and can be powered using mains voltage of 100-240 VAC, 50-60 Hz. Main
power fuses (F3A, 250 V, 5x20mm) are located behind a small black plastic cover between main switch and
the power main plug at the back of the instrument. The Xdrop™ instrument must be installed on a flat surface
where access to the main power outlet is not restricted. The instrument is not to be used against the
manufacturers instructions. Failing to comply with the requirements can result in potential hazards to the
instrument and the user.

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Icon

Meaning

Instrument safety warnings

The following warning labels refer directly to the safe use of the Xdrop™ instrument.

Warning about the risk of harm to body or equipment. Operating the

Xdrop™ before reading this manual can constitute a personal injury

hazard. Only qualified laboratory personnel should operate this

instrument.

Warning about the risk of harm to body or equipment from electrical

shock. Do not attempt to repair or remove the outer case of this

instrument, power supply, or other accessories. If you open these

instruments, you put yourself at risk for electrical shock and void

your warranty. All repairs must be done by an authorized repair

service.

Warning about the risk of harm to hands and fingers. Always keep

hands and fingers away from the instrument when the drawer is in

motion.

Intended use and intended users

The Xdrop™ instrument is intended for use by trained laboratory personnel in a clean laboratory environment
for DNA sample preparation using droplet microfluidic technology.

Transportation and storage

Always transport the instrument in the original Samplix box. Before starting up the instrument, let it stay in
room temperature for at least 2 hours.

Maintenance and cleaning

If the instrument is shipped back to Samplix for maintenance, please make sure that the outer surfaces are
cleaned using a cloth and 70 % ethanol.

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Xdrop™ Installation and Set-Up Quick Guide

1. Place the transport box on a flat surface.

2. Flip out the four lock twisters and turn them counterclockwise to unlock the lid of the
transportation box.

3. Remove the lid to gain access to the instrument.

4. Slide a hand into the box on each side of the instrument and lift it out of the box.

Tip: If required, gently lift the back of the instrument 10-15 cm and place it against the
foam/padding at the back. This should allow you to get a good grip placing both hands under
the instrument.

5. Lift out the instrument and place it on a flat horizontal surface.

6. Leave the instrument unused for at least 2 hours.

Note: Leaving the instrument at ambient temperatures allows the instrument to equilibrate and
will reduce the risk of instrument failure.

7. Attach the power cord to the back of the instrument. Note: Use the included power cable only!

8. Plug the power cable into an appropriate power outlet.

9. Turn the main switch at the back of the instrument to the ”|” position.

10. Press the Power button at the front to power up the instrument.

Note: Power button needs to be fully pressed for the instrument to start up.

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Name

Cat. No.

Xdrop™ Instrument

IN00100

dPCR cartridge

CA10100

dPCR gasket

GA10100

Storage film

FI00100

dPCR kit

Break colour ● (4⁰C)

Name

Cat. No.

Xdrop™ Instrument

IN00100

dMDA cartridge

CA20100

dMDA kit

Required items for Xdrop™

dPCR

RE10100 Part 1

(store at -20°C)

dPCR mix (2x) ●
Droplet dye ●

Part 2

(store at -20°C)

dPCR buffer (2x) ●

dMDA

dMDA holder HO10100

dMDA gasket GA20100

Storage film FI00100

RE20300 Part 1

(store at -20°C)

dMDA mix (5x) ●
dMDA enzyme ○

Part 2

(store at RT and 4⁰C)

dMDA oil ● (RT)
Break Solution ● (RT)
Break colour ● (4⁰C)

Part 3

(store at RT and 4⁰C)

dPCR oil ● (RT)
Break Solution ● (RT)

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Name

Cat. No.

Cell sorter control kit

CO10100

Primer test PCR kit

RE10200

Suggested Samplix items

Positive control

primer kit

CO10200

Part 1

(store at -20°C)

Droplet dye ●
dPCR buffer (2x) ●

Part 1

(store at -20°C)

dPCR control primers ●
Enrichment validation primers ●
Positive control DNA ●

Part 1

(store at -20°C)

dPCR mix (2X) ●
qPCR dye (20x) ●

Part 2

(store at 4°C)

Control droplets ○

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Equipment

Equipment and reagents for Xdrop™ enrichment and amplification supplied by the user

In addition to required and suggested Samplix products, the following items are required.

Cell sorter instrument

Real-time PCR cycler

Thermal cycler with slow ramping function

Instrument for nucleic acid quantification e.g. Qubit™, Bioanalyzer™, TapeStation™, FEMTO Pulse™ or

similar.

LAF (Laminar Air Flow) hood

Microcentrifuge

Vortex

Pipette set from P2 to P1000

Freezing blocks for both PCR tubes and microcentrifuge tubes

Consumables

Nuclease free PCR tubes and microcentrifuge tubes

Eppendorf DNA LoBind tubes 1,5 ml tubes (Catalog No. 0030108051)

Filtered pipette tips

Wide bore pipette tips for P200 pipette outer diameter of tip: 1 mm – 1,9 mm

Reagents

Nuclease-free water

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Chapter 2: Droplet PCR Reaction Design
for Target Selection

The Xdrop™ technology requires a simple assay design with the following components:

• A high molecular weight DNA sample ( > 30 kb, depending on the assay) of high purity. Calculate the

required amount of input DNA needed based on the desired enrichment and desired amount of
output DNA using the online DNA input calculator at samplix.com

• One droplet PCR (dPCR) primer pair for enrichment. Please see the design guidelines below and

use the online primer design tool at samplix.com

• One quantitative PCR (qPCR) QC primer pair for validation of Xdrop™ DNA enrichment. Please see

the design guidelines below and use the online primer design tool at samplix.com

Target sequence

The Xdrop™ technology allows targeted enrichment by selection and amplification of a genomic region of
interest without the need for long-range PCR. The target DNA of interest can contain repeat regions, GC-rich
regions or other regions that are otherwise difficult to amplify. Specific primers amplifying a short fragment
of 120-160 bp (ID sequence), within the target or in flanking regions, are used for capturing the kilobases­long region of interest. The Xdrop™ technology compartmentalizes the amplification reaction in small
droplets and makes use of a highly processive DNA polymerase to enable amplification of almost all regions
of the genome.

The length of the enriched target DNA will depend on the length of the input DNA. Consider using high
molecular weight DNA as input with a DNA fragment size > 30 kb and of high purity. Calculate the optimal
amount of input template DNA using the online sample input calculation tool at samplix.com

DNA sample preparation

When purifying the DNA sample, use a method that maintains the integrity and the purity of the DNA. Take
all required measures to avoid contaminations. The Xdrop™ enrichment technology can be affected by
contamination of the DNA sample by RNA, proteins, carbohydrates, salt and phenol among others. Purify the
DNA to the same quality as required for long read sequencing.

Calculate the optimal amount of input template DNA using the online enrichment calculation tool at

samplix.com

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dPCR primers

qPCR QC primers

Amplicon length

120-160 bp

80-120 bp

Melting temperature

~ 60°C

~ 60°C

Primer design guidelines

The Xdrop™ enrichment technology relies on carefully designed and highly specific PCR primer pairs. Two
sets of non-overlapping PCR primer pairs are required. The first one is called dPCR primer set and is
responsible for creating a fluorescent signal used for target selection. The second set of PCR primers, called
the qPCR QC primer set, is used to validate the assay and quantify the number of target fragments in the
pool of enriched fragments.
If the target spans over a region of 50 kb or longer, a combination of two or more sets of dPCR/qPCR primers
are recommended.

Help for designing primers can be found in the online primer design tool at samplix.com

General guidelines for primer design

Apart from the dPCR primer pair, a qPCR QC primer pair is required for validation and calculation of the
enrichment.

Note: The qPCR QC primer pair must be different from the enrichment dPCR primer pair and placed within
2 kb distance from it. The qPCR QC and dPCR amplicons must not overlap.

The risk of false-negative enrichment prediction increases if the validation qPCR assay is placed further

away from the dPCR assay.

The following guidelines apply to both dPCR and qPCR QC primer pairs:

• Avoid primer pairs with more than 2°C difference in melting temperature between forward and

reverse primer.

• Avoid placing primers in low complexity regions.

• Primers need to be specific. Avoid primer pairs that amplify sequences not related to the target

sequence.

• Follow the general recommendations for PCR primer design: avoid self-complementarity, stable

secondary structures, hairpins etc.

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dPCR reaction mix

1 reaction

H2O (molecular grade)

7,2 μl

dPCR mix (2x) ●

10 μl

Primer Forward 10 µM

0,4 μl

Total mix

19 μl

Template DNA

1 μl

Total volume

20 µl

Temperature

Duration

Cycles

30°C

5 sec

1x

94°C

2 min

94°C

3 sec

Annealing temp.

30 sec

Data acquisition*

-

Melting curve 70-

90°C

1X

Primer testing

For every new target and prior to generating droplets, both the dPCR enrichment primers and the qPCR QC
primers must be optimized preferably by qPCR using your sample DNA and Samplix dPCR reagents ● and
qPCR dye ●. For this assay, include a negative control with no template and at least three different
concentrations of your template (or a biological replicate) in the range of the amount of input DNA suggested
by the online sample calculation tool at samplix.com (see Fig. 2.1).

Use the Samplix primer test PCR kit (Cat. No. RE10200) that includes Samplix dPCR mix (2X) ● and Samplix
qPCR dye ● to verify the primers and reaction efficiency.

The primer test reagents are optimized for the following reaction mix using Samplix Primer test PCR kit (Cat.
No. RE10200):

Primer Reverse 10 µM 0,4 μl

qPCR dye (20x) ●

And the following qPCR program:

*Follow manufacturer’s protocol for data acquisition program.

Note: The annealing temperature needs to be adjusted to the specific primer sets tested
Note: qPCR QC primer can be tested either in the Samplix dPCR reagents or in your preferred qPCR reagents

1 μl

40x

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We recommend running a melting curve analysis with the template DNA, dPCR primers, and Samplix reagents
to check for the presence of alternative amplicons and primer-dimers. Consider running a temperature
gradient to determine the optimal annealing temperature.

The supplied qPCR dye (20x) ● maximum values for excitation and emission are approximately 497 nm and
520 nm respectively (SYBR

Calculate the PCR efficiency using the Ct values as input with the formula: (10^(-1/slope)-1) *100

Make sure that your designed primer pairs have an efficiency between 90-110 % and that the suggested DNA
input amount yields a Ct value below 30.

TM

green settings). Make sure these values are selected in your qPCR instrument.

Fig. 2.1. Calculate PCR efficiency with at least three different concentrations of input DNA using the Samplix

Primer test PCR kit (Cat. No. RE10200) and your designed primers. Left: Amplification plot of three DNA
concentrations. Right: Standard curve and calculations of PCR efficiency.

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