Samplix Xdrop User manual

Xdrop™ manual
Droplet PCR (
dPCR)
User manual v. 1.0 released 26 Aug. 2019

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Table of Contents
Chapter 1: Xdrop
TM
at a Glance page 03
Targeted enrichment overview
Xdrop™ instrument overview
Required items for Xdrop™ dPCR
Suggested Samplix products
Equipment and reagents for Xdrop™ supplied by the user
Chapter 2: dPCR – General Considerations page 09
dPCR assay design
Target sequence
Primer design guidelines
DNA sample preparation
Primer optimization
Chapter 3: dPCR Setup page 12
Preparations for dPCR
Setup of dPCR reaction
Positive control dPCR reaction
Prepare dPCR cartridge
Collect generated droplets
Chapter 4: Single DNA Molecule Detection and Sorting of Droplets page 20
Requirements for flow cytometer analyzer and cell sorter
Notes to operator
Preparation of droplets for flow cytometry
Flow cytometry analysis of dPCR droplets
Set up flow cytometry with Cell sorter control droplets
Sorting of PCR positive dPCR droplets
Contact:
Samplix ApS
Mileparken 28
DK – 2730 Herlev
www.samplix.com
Mail: [email protected]
For research use only, not for use in diagnostic procedures.
Copyright 2019. 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™ technology introduces a new approach for genomic analysis as the technology enables targeted
enrichment of genomic regions in droplets. The Xdrop™ technology offers sensitive and unbiased PCR-free sample
enrichment and general amplification prior to downstream analysis e.g. next generation sequencing.
Using the Xdrop™ instrument, cartridges, and reagents, sample DNA is partitioned into millions of picolitre size highly
stable droplets. The Xdrop™ droplet PCR (dPCR) droplets are suitable for standard PCR cycling, flow cytometry analysis
and sorting.
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. Droplets containing the target DNA molecules are
identified by a 120-160 base pair targeted PCR specific to a sequence within or adjacent to the region of interest.
Positive droplets are identified by their fluorescence and physically separated from negative droplets by use of a
standard cell sorter. The result is an enrichment of long single molecules comprising tens of kilobases of DNA
information.
For downstream universal amplification of the single molecules, Samplix has developed a proprietary technology by
which the high molecular weight DNA molecules are partitioned into thousands of 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 DNA sequencing.

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Xdrop™ instrument overview
The Xdrop™ droplet generator is compatible with Samplix dPCR cartridges for production of dPCR droplets
and Samplix dMDA cartridges for the generation of 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.
The Xdrop
™
droplet generator is composed of the following parts (see figure below):

Drawer – holds the dPCR or dMDA cartridge.

Touch screen – provides the means to control the droplet generator with gloved or un-gloved
hands.
 USB port on the back of the instrument – connects to a USB flash drive for troubleshooting, saving log files,
and for updating instrument firmware.

Status LED – Green when in standby and operating and yellow-green when opening and closing the
drawer.

Air vents on the back of the instrument – for ventilation.

A start button on the front of the instrument.

A hardware switch on the back of the instrument.

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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
Support
To find technical support, contact the technical support team at [email protected]
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.

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Instrument safety warnings
The following warning labels refer directly to the safe use of the droplet generator.
Icon Meaning
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.
Never remove the outer case of an Xdrop ™ instrument. This may cause
you to receive an electrical shock.
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 from mixed DNA samples 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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Required items for Xdrop™ dPCR
Xdrop™ Instrument (Cat. No. IN00100)
dPCR cartridge (Cat. No. CA10100)
dPCR gasket (Cat. No. GA10100)
Storage film (Cat. No. FI00100)
dPCR kit (Cat. No. RE10100)
dPCR kit part 1 (store at -20°C)
dPCR mix (2x) ●
Droplet dye ●
dPCR kit part 2 (store at -20°C)
dPCR buffer (2x) ●
dPCR kit part 3 (store at room temperature)
dPCR oil ●
Suggested Samplix products
Cell sorter control kit (Cat. No. CO10100)
Cell sorter control kit part 1 (store at -20°C)
Droplet dye ●
dPCR buffer (2x) ●
Cell sorter control kit part 2 (store at 4°C)
Control droplets ○
Positive control primer kit (store at -20°C) (Cat. No. CO10200)
dPCR control primers ●
Enrichment validation primers ●
Positive control DNA ●
Primer test PCR kit (store at -20°C) (Cat. No. RE10200)
dPCR mix (2X) ●
qPCR dye (20x) ●

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Equipment and reagents for Xdrop™ enrichment and amplification supplied by the user
In addition to required and suggested Samplix products, the following items are also suggested.
Thermal cycler
Real-time PCR cycler
LAF (Laminar Air Flow) hood
Flow cytometry analyser / cell sorter
Quantification of nucleic acids – Nanodrop, Qubit, Quantus, Bioanalyzer, Tapestation or similar
Microcentrifuge
Vortex
Freezing blocks for both PCR tubes and microcentrifuge tubes
Nuclease-free water
Nuclease-free tubes and filter pipette tips
Wide bore pipette tips (P200 Pipette, Orifice size: 1.5 mm)
PCR tubes
1.5 ml LoBind tubes

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Chapter 2: droplet PCR – General
Considerations
dPCR assay design
The Xdrop™ technology requires a simple assay design with the following components:
 A DNA sample 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 sample input calculation tool 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) 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 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 100 bp is used for capturing the 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

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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; one set of PCR primer pairs, called the dPCR set, is responsible for creating
a fluorescent signal used for target enrichment. The second set of PCR primers, called the qPCR set, is used to validate
the assay and quantify the number of target fragments in the pool of enriched fragments.
Help for designing primers can be found in the online primer design tool at samplix.com
General design guidelines for dPCR primer pairs are as follows:
 Amplicon length between 120 and 160 base pairs.
 Melting temperature around 60°C.
 Avoid primer pairs with more than 2°C difference in melting temperature between forward and reverse
primer.
 Avoid placing primers in low complexity regions on the target.
 Primers need to be specific. Avoid primer pairs that amplify sequences not related to the target sequence.
 Follow the general considerations for PCR primer pairs, avoid self-complementarity, stable secondary
structures, etc.
The second primer pair required is for validation and calculation of the enrichment. NB: The second primer pair (the
qPCR primer pair) must be different from the enrichment dPCR primer pair and amplicons must not overlap.
General design guidelines for qPCR primer pairs for validation of enrichment are as follows:
 Amplicon length between 80-120 base pairs.
 Melting temperature around 60°C.
 Avoid primer pairs with more than 2°C difference in melting temperature between the two primers.
 Place the amplicon within 2 kb of the droplet primer pair without overlapping the dPCR amplicon. The risk of
false-negative validation increases if the validation qPCR assay is placed further from the dPCR assay.
 Follow the general considerations for PCR primer pairs, avoid self-complementarity, stable secondary
structures, etc.
DNA sample preparation
When purifying the DNA sample, use a method that maintains the integrity of the DNA fragments while giving pure DNA
without 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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Primer optimization
The design and location of the primers used must be optimal for the assay. Optimize the dPCR reaction preferably by
qPCR using your sample DNA as a template. Include a negative control with no template and at least three different
concentrations of your template in the range of the amount of input DNA for the dPCR assay suggested by the online
sample calculation tool at samplix.com.
It is advised to run a melting curve analysis with the template DNA, dPCR primers designed and Samplix reagents to
check for the presence of alternative amplicons and primer-dimers. Consider running a temperature gradient to
determine the best annealing temperature.
Use the Samplix primer test PCR kit (Cat. No. RE10200) with Samplix dPCR mix (2X) ● and Samplix qPCR dye ● to verify
the primers and reaction efficiency.
To calculate the PCR efficiency, run a qPCR with a standard curve with at least three different amounts of input DNA
using the Samplix dPCR mix and your designed primer pairs (Fig. 2.1).
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 %.
Fig. 2.1.
Standard curve of the number of cycles versus fluorescent signal with three different amounts of input DNA for
calculation of PCR efficiency. To determine the PCR efficiency of the dPCR reaction, run a qPCR reaction using Samplix
dPCR mix (2X) ● and your designed primer pair with increasing amounts of input DNA.

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Chapter 3: dPCR Setup
Preparations for dPCR
Use the Samplix Primer test PCR kit (Cat. No. RE10200) to determine the optimal primer concentration and annealing
temp for the dPCR reaction in the Samplix dPCR mix (2X) ● as described in the previous chapter (Chapter 2: dPCR –
General Considerations). NB: Do not use any other reagents instead of Samplix dPCR kit (Cat. No. RE10100) for Xdrop™
dPCR droplet production as this may compromise droplet production, droplet stability, and downstream enrichment.
Samplix provides a Positive control kit (Cat. No. CO10200) that contains DNA template and two primer pairs; one for
dPCR and one for validation qPCR after dMDA. See instructions next page.
Setup of dPCR reaction
1. Prepare the dPCR reaction mix after the table below.
2. Prepare mix for all reactions (40 μl/reaction total). Keep the reagents and the dPCR reaction mix on ice.
3. Prepare dilutions of the correct amount of template DNA. Calculate the optimal amount of input template DNA
using the online sample calculation tool samplix.com
4. Add 2 μl of template DNA dilution to 38 μl dPCR reaction mix to get 40 μl/reaction total.
5. Keep the reaction mix at 4°C until loading on dPCR cartridge followed by immediate insertion of the dPCR cartridge
on the Xdrop™ instrument.
dPCR reaction mix 1X
H2O (molecular grade) 17,2 μl
dPCR mix (2x) ● 20 μl
Primer Forward 10 µM 0,4 μl
Primer Reverse 10 µM 0,4 μl
Total mix 38 μl
Template DNA 2 μl

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Optional: Positive control dPCR reaction (Cat. No. CO10200)
The Positive control kit (Cat. No. CO10200) is designed as a positive control assay to use together with the dPCR kit (Cat.
No. RE10100) and dMDA kit (Cat. No. 20100) for the full Xdrop™ workflow; dPCR droplet production, sorting and
enrichment.
 Set up dPCR reaction mix using dPCR control primers ● and Positive control DNA ● as described below.
 Generate dPCR droplets in the Xdrop™ droplet generator as described from p. 14.
 Run the produced dPCR droplets with the PCR program described p. 19 using an annealing temperature of
60°C.
 Sort positive fluorescent dPCR droplets (see Chapter 4: Single DNA Molecule Detection and Sorting of
Droplets).
 Amplify sorted DNA material by dMDA, see XdropTM Manual Droplet MDA (dMDA).
 Validate enrichment as described in XdropTM Manual Droplet MDA (dMDA) using Enrichment validation primers ●
Set up the positive control dPCR reaction as detailed below:
1. Prepare the positive control dPCR reaction mix after the table below using dPCR control primers ● and Positive
control DNA ●
2. Prepare mix for all reactions (40 μl/reaction total). Keep the reagents and the dPCR reaction mix on ice.
3. Use the dPCR control primers ● for the dPCR reaction mix.
4. The Positive control DNA ● is already at the correct concentration and should not be diluted further.
5. Keep the reaction mix at 4°C until loading on dPCR cartridge followed by immediate insertion of the dPCR cartridge
on the Xdrop™ instrument.
Control dPCR reaction mix 1X
H2O (molecular grade) 17,2 μl
dPCR mix (2x) ● 20 μl
dPCR control primers ●
(Forward and reverse primer)
0,8 μl
Positive control DNA ● 2 μl
Total mix 38 μl
6. Prepare the dPCR cartridge as described below.

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Prepare dPCR cartridge
1. Dilute the 2X dPCR buffer ● to 1X with molecular grade water. NB: Dilute an entire vial of dPCR buffer.
Precipitate potentially forming in the 2x buffer will dissolve when diluted to 1x. Make sure to mix well by
vortexing 10 seconds and inverting the tube >20 times. Use leftover 1x dPCR buffer for sorting of droplets.
2. Unpack the cartridge from the plastic bag and place it on a clean flat surface in a LAF (Laminar Air Flow) hood
or a similar dust free environment. The layout of the cartridge is depicted in Fig. 3.1.
3. Handle the cartridge as follows:
 Always use gloves when handling the cartridge.
 Hold the cartridge by its sides.
 Do not touch any of the input wells or droplet exit well.
 Do not touch the black microfluidic chip on the back of the cartridge.
 Save the cartridge storage bag for later storage of the cartridge.
4. Be careful not to use the same line more than once as this will disrupt droplet production. To avoid using the
same line more than once mark the storage plastic bag or the cartridge directly with a permanent marker
once a line has been used.
Fig. 3.1. The dPCR cartridge. Wells are marked A, B, C, and D. Lines are marked 1 – 8.
5. Load the cartridge with reagents in a LAF (Laminar Air Flow) hood or a similar clean, dust free environment.
Keep the dPCR reaction mix at 4°C until loading. NB: it is important to load the reagents on the cartridge in the
order described below.

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Fig. 3.2. Loading of the dPCR cartridge. Left: Avoid introducing air bubbles by pipetting on the side of the wells. Right: dPCR cartridge
top view with wells marked.
6. Load 300 μl 1x dPCR buffer in the first well (#A). It is important to load the cartridge in the order described
here and avoid air bubbles by pipetting carefully on the side of the well (Fig. 3.2).
7. Load 40 μl 1x dPCR buffer to the shelf of the outlet well #D (Fig. 3.2).
8. Load 40 μl dPCR reaction mix including primers and sample DNA into well #C.
9. Load 100 μl dPCR oil ● into well #B.
10. Notice that the order of loading is: 300 μl 1x dPCR buffer in the well #A.
40 μl 1x dPCR buffer to the shelf #D.
40 μl 1x dPCR reaction mix in well #C.
100 μl dPCR oil ● in well #B.
11. Add a gasket to the top of the cartridge. Orient the gasket to the cartridge using the angled corner. Attach to
the pins first and then to the hooks (Fig. 3.3).
Fig. 3.3. Cover cartridge with the gasket by orienting it correctly (angled corner on the gasket to
angled corner on cartridge), then attach first the pins then pull gently to attach hooks.

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Before powering up the instrument please make sure that the main power switch is in the “1” position. The main switch
is located at the back of the instrument. Power up the instrument by pushing the Start/Stop button at the front. The
instrument will start initializing and will shift to “Welcome screen”.
12. Push “open” on the instrument touch screen to eject the drawer (Fig. 3.4).
Fig. 3.4. The Xdrop™ instrument “welcome” screen, press “open” to eject the drawer.
13. When the “open” or “next” button has been pressed, the screen shifts to “Please place cartridge” and “close”.
Make sure that the cartridge is correctly positioned into the drawer (Fig. 3.5) as it may otherwise cause
damage to the instrument. Once the cartridge is correctly inserted, press “close” to retract the drawer into the
instrument.
Fig. 3.5. The Xdrop™ instrument with a correctly inserted dPCR cartridge. Be very careful to place the cartridge
correct as the instrument may otherwise damage the cartridge.

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14. Once the drawer is fully closed, press “next” on the touch screen.
15. The droplet generator can operate with either dPCR or dMDA cartridges. Use the dPCR cartridge with the
“dPCR” option on the touch screen (Fig. 3.6.).
Fig. 3.6. The Xdrop™ instrument “Select cartridge” screen, select dPCR cartridge.
16. The lines to be processed are selected by pressing the corresponding numbers 1-8 on the screen. When
selected, buttons turn green (green = selected & blue = not selected) (Fig. 3.7).
Fig. 3.7. Selecting the lines to be used. Selected channels will be indicated by green buttons (here: 2, 4, 6 and 8). Blue
buttons indicate channels not yet selected.
17. Press “run”.

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The instrument will now build up pressure. This process can take up to 4 minutes.
Once the desired pressures have been built up the message “Making your droplets” and the remaining run time are
displayed on the screen (Fig. 3.8). The Xdrop™ instrument will produce droplets in 40 minutes.
Fig. 3.8. Touch screen image while producing droplets.
18. When droplet production has been completed, the screen will change to “Your droplets are ready”.
(Optional: To save the Log file from this production, insert a FAT formatted USB stick into the USB port at the back of
the instrument. 1-2 seconds after having inserted the USB, a “Save Log” button will appear on the touch screen. Push
the button and enter an appropriate name for your log file).
19. Press “Open” to eject the cartridge.
20. Remove the cartridge from the instrument and place it in a LAF bench.
21. Press “Close” to make the instrument retract the drawer back into its closed position.
22. Press “Finish” to return to the Welcome screen.
23. Shut down the instrument after a completed droplet production to avoid damage to the instrument. If the
instrument is not to be used for several hours, it should preferably be turned off. To turn the instrument off,
push the ON/OFF button at the front to power off the instrument to initiate the automatic shutdown
procedure.
Please note! Each production line is single use and will not function properly if an attempt to re-
use already processed lines is carried out. Furthermore, attempting to reuse the cartridge will
increase the risk of cross-contamination of samples.

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Collect generated droplets
24. After finalized droplet production, confirm that dPCR droplets have been produced. dPCR droplets will sink to
the bottom of the collection well and form a white layer with a clear buffer phase on top.
25. If droplets are visible, collect droplets from the Exit-well (#D).
26. Optional: 200 μl dPCR-buffer (surplus in the dPCR-buffer well #A) can be used to “wash” residual droplets from
the shelf inside the exit well - where droplets are entering the well #D.
27. Remove excess liquid in wells #A and #B before storage of the cartridge.
28. Store the cartridge at room temperature in the provided bag with the gasket placed on top of the cartridge.
A total volume of droplets and buffer around 300-400 μl is expected to be collected after droplet production.
29. Dispense the droplets into four PCR-tube aliquots (of approximately 80-90 μl). NB: Please note that dPCR
droplets sediment rapidly during handling. To ensure equal distribution into the aliquots, be sure to mix gently
by pipetting up and down between each pipetting step.
30. Place the PCR-tubes in a thermocycler and run the following program. A temperature ramp rate of 1,5°C/sec
or less is suggested.
Temperature
Duration
Cycles
30°C
5 sec
1x
94°C 2 min Slow ramp
1,5°C/sec
94°C 3 sec Slow ramp
1,5°C/sec
40x
Annealing temp. 30 sec
24°C 30 sec 1x
Store the droplets at 4°C after PCR.
31. Continue with dPCR droplet sorting (see Chapter 4) within 24 hours to maintain the integrity of the DNA
fragments. dPCR droplets can be stored after PCR at 4°C for up to 24 h if proceeding with DNA enrichment.
dPCR droplets are stable at 4°C up to 2 months, however, storage longer than 24h will compromise DNA
quality and enrichment and should be avoided.
Good pause point: Store dPCR droplets after PCR at 4°C for up to 24 hours.

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Chapter 4: Single DNA Molecule
Detection and Sorting of Droplets
Requirements for flow cytometer analyzer and cell sorter
 A 488 nm (blue) laser.
 Optical configuration detecting fluorophores excited at 488 nm such as FITC, GFP, and PE.
 100 μm nozzle tip/sorting chip minimum.
 Sheath pressure at a maximum of 24 psi.
 Sample line should be positioned at the bottom of the sample tube.
Notes to operator
 dPCR droplets are large, therefore the correct events are high in Forward Scatter (FSC) and Side Scatter (SSC)
(see fig. 4.1).
 Smaller events and events with low SSC represent pure oil droplets and should be gated out.
 dPCR droplets are stable and relatively heavy. It could take 5-10 min before the dPCR droplets reach the point
of interrogation and appear on the plot.
 dPCR droplets do not clump together and a duplet discrimination plot is not required.
 Positive fluorescent droplets are likely to be very rare (possibly less than 0,02% of total dPCR droplets).
Therefore, a positive population can be difficult to identify. Make sure the live plot of fluorescence shows at
least 100.000 events (see fig. 4.2) A histogram plot is not recommended.
Preparation of droplets for flow cytometry
1. Remove tubes with dPCR droplets from the PCR machine.
2. Make sure the 2x dPCR buffer ● is diluted with molecular grade water to 1x. Mix well by vortexing 10 seconds
and inverting the tube at least 20 times.
3. Stain droplets with Droplet dye ● follows:
 Prepare flow cytometry buffer by adding 1ml 1x dPCR buffer to a flow cytometry tube (tubes
depend on flow cytometer instrument).
 Spin down Droplet dye ● 1-2 minutes.
 Add 10 μl Droplet dye ● into the flow cytometry tube with dPCR buffer. Mix gently to
dissolve the dye in the dPCR-buffer.
 Remove supernatant from PCR tube leaving dPCR droplets undisturbed at the bottom.
 Use 200 μl buffer from flow cytometry tube to transfer all droplets from the PCR tubes.
 Leave at room temperature protected from light for 5 min to stain droplets.
4. Load tube containing the dPCR droplets on the flow cytometer and start analysis or sorting.