Xdrop™ manual User manual v. 2.0 released 14 Feb.
Table of Contents Chapter 1: XdropTM at a Glance Targeted enrichment overview Workflow overview Xdrop™ instrument overview Required items for Xdrop™ Suggested Samplix products Equipment and reagents for Xdrop™ supplied by the user page 3 Chapter 2: Droplet PCR Reaction Design for Target Selection Target sequence Primer design guidelines Primer Testing page 12 Chapter 3: Droplet PCR (dPCR) Preparations for dPCR Setup of dPCR reaction Optional: Positive control dPCR reaction Prepare dPCR cartridge Collect
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.
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.
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 of the following parts (see figure below): - A start button on the front of the instrument.
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 support@samplix.com 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.
Instrument safety warnings The following warning labels refer directly to the safe use of the Xdrop™ instrument. 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.
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.
Required items for Xdrop™ dPCR Name Cat. No. Xdrop™ Instrument IN00100 dPCR cartridge CA10100 dPCR gasket GA10100 Storage film FI00100 dPCR kit RE10100 Part 1 Part 2 Part 3 (store at -20°C) (store at -20°C) (store at RT and 4⁰C) Part 1 Part 2 (store at -20°C) (store at RT and 4⁰C) dPCR mix (2x) ● Droplet dye ● dPCR buffer (2x) ● dMDA Name Cat. No.
Suggested Samplix items Name Cat. No.
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. Equipment 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.
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.
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.
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).
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 (SYBRTM green settings). Make sure these values are selected in your qPCR instrument.
Chapter 3: Droplet PCR (dPCR) Preparations for dPCR Use the Samplix Primer test PCR kit (Cat. No. RE10200) to determine the optimal primer concentration and annealing temperature for the dPCR reaction as described in the previous chapter (Chapter 2: Droplet PCR Reaction Design for Target Selection). NOTE: 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.
Optional: Positive control dPCR reaction (Cat. No. CO10200) Note that it is possible to include the Positive control kit (Cat. No. CO10200) provided by Samplix into the dPCR reaction. The kit includes both Positive control DNA ● and dPCR control primers ● (already mixed) with an annealing temperature of 60°C. See table below.
4. Be careful not to use the same lane more than once as this will disrupt droplet production. To avoid using the same lane more than once mark the storage plastic bag or the cartridge directly with a permanent marker once a lane has been used. 5. Load the cartridge with reagents in a LAF hood or a similar clean, dust free environment. Allow the reaction mix to reach room temperature before loading on the dPCR cartridge followed by immediate insertion on the Xdrop™ instrument.
Well #D shelf Fig. 3.2. Cross section of the dPCR cartridge. Notice the exact location of the shelf in well #D, where 40 μl of 1x dPCR buffer ● are added. When loading the dPCR cartridge, avoid introducing air bubbles by pipetting on the side wall of the wells. 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. 7.
10. Add a white rubber 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 T-hooks (Fig. 3.3). First insert pins Then insert T-hooks Fig. 3.3. Cover the cartridge with the white rubber 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 T-hooks.
12. When the “Open” or “Next” button have been pressed, the screen shifts to “Please insert/remove 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.
Fig. 3.6. The Xdrop™ instrument “Select cartridge type” screen, select dPCR cartridge. 15. The lanes to be processed are selected by pressing the corresponding numbers 1-8 on the screen. When selected, buttons turn green (green = selected and blue = not selected) (Fig. 3.7). Fig. 3.7. Selecting the lanes to be used. Selected channels will be indicated by green buttons (here: 1 to 4). Blue buttons indicate channels not yet selected. 16. Press “Run”. The instrument will now build up pressure.
Once the optimal pressures have been reached, the message “Making your droplets” and the remaining run time are displayed on the screen (Fig. 3.8). The Xdrop™ instrument will produce double emulsion dPCR droplets in approximately 40 minutes. Fig. 3.8. Touch screen image while producing droplets. 17. When droplet production has been completed, the screen will change to “Your droplets are ready”. 18. Press “Open” to eject the cartridge. 19. Remove the cartridge from the instrument and place it in a LAF hood.
Collect generated droplets 23. After 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. 24. Collect droplets from the collection well (#D) into a 0,5 ml or 1,5 ml tube. Use tips that minimize binding of droplets to the side of the tip. 25.
29. Place the PCR tubes in a thermal cycler and run the program described below. A block temperature ramp rate of 0,5°C/sec is recommended, with a total duration of the program of ~2 hours. This is to ensure slow temperature changes, allowing the reagents inside the droplets to reach the desired temperature. 30. Set the lid temperature to 105°C. Temperature Duration Ramp rate Cycles 30°C 5 sec - 1x 94°C 2 min block ramp 0,5°C/sec 94°C 3 sec block ramp 0,5°C/sec Annealing temp.
Chapter 4: Single DNA Molecule Detection and Sorting of Droplets Double emulsion droplets generated with the dPCR cartridge can be sorted and collected in a standard cell sorter, capturing the DNA of interest. In this step, the positive dPCR droplets containing the region of interest are identified and separated from the negative droplets using the fluorescent signal provided by the dPCR amplification of the ID sequence (see Chapter 3). Requirements for cell sorter • • • • A 488 nm (blue) laser.
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 for 10 seconds or inverting the tube at least 20 times. 3. Stain droplets with Droplet dye ● as 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 at 5000 rpm ● 2 minutes.
Flow cytometry analysis and sorting of dPCR droplets 7. Identify the dPCR droplet population on a plot of FSC (height) versus SSC (height) (Fig. 4.1). dPCR droplets are higher in side scatter (SSC) than oil droplets. Note that it could take a few minutes before the heavy dPCR droplets are analyzed and visible in the plot. Fig. 4.1. Identify dPCR droplets on a plot showing forward scatter (FSC) versus side scatter (SSC) or back scatter (BSC).
Fig. 4.2. Identify positive fluorescent population versus side scatter. The positive population is green, the negative population blue and the oil droplets grey in this figure. Set the software to show at least 100.000 events in the plot. 9. Set the gates as detailed above (Fig. 4.2), taking care to draw a strict gate around the population of interest. Note: be aware of potential drift in fluorescence over time and be prepared to move the sorting gate during the sorting if required. 10.
Optional: Set up flow cytometry with Cell sorter control kit (Cat. No. CO10100) To easily set up flow cytometry of dPCR droplets, use the Samplix Cell sorter control kit (Cat. No. CO10100). This kit consists of ready-made dPCR droplets with a defined and large population of positive droplets. The kit allows you to establish the settings for dPCR droplet sorting. 1. Make sure the 2x dPCR buffer ● is diluted with molecular grade water to 1x.
Chapter 5: Multiple Displacement Amplification in droplets (dMDA) Break sorted droplets (if applicable) After sorting, keep the sorted dPCR droplets at 4°C and proceed immediately to dMDA amplification. Do not store sorted dPCR droplets longer than 8 hours as this may lead to DNA degradation. If amplifying already purified DNA, continue directly to “Set up dMDA reaction”, described on the next page.
Set up dMDA reaction Note: Do not use any other reagents than Samplix dMDA kit (Cat. No. RE20300) for Xdrop™ dMDA droplet production as this may compromise droplet production, droplet stability, and downstream enrichment. Thaw and keep all reagents at 4°C or in a cooling block while setting up the reactions, except the oil that should be kept at RT. The MDA reaction is very susceptible to contamination. Make sure to avoid DNA contaminations of any kind.
Load the dMDA cartridge The dMDA reaction takes place inside droplets formed in the dMDA cartridge inserted in the holder (Fig. 5.2). The cartridge must be sealed with a gasket on top during droplet production. Load samples in a clean LAF hood. Note: The dMDA holder should be re-used for all the following runs. dMDA holder dMDA cartridge Inlet well Catridge handle Collection well dMDA oil Sample mix Fig. 5.2. Left: dMDA holder.
• • Be cautious to avoid DNA contamination at all times. Store the cartridge (if partially used) covered by protective storage film in a clean, sealed plastic bag. 3. Be careful not to use the same sample lane more than once as this will disrupt droplet production and lead to contamination of your sample. In order to avoid using the same lane more than once, mark the storage plastic bag with a permanent marker once a lane has been used. Fig. 5.3.
Load sample mix here Outer diameter 1- 1,9 mm Fig. 5.4. Injection of the sample in the inlet well. From left to right: Cartridge inlet where to load the sample mix: the wide bore pipette tip should enter the hole in the pointy end of the tear shaped inlet well. Correct positioning of the wide bore pipette tip. Incorrect positioning of the wide bore pipette tip. Outer diameter of the wide bore pipette tip (1-1,9 mm). Picture of dMDA cartridge loading of the sample mix.
9. Add 75 μl dMDA oil ● to the side of the inlet well allowing it to flow gently into the reservoir in the loaded lane(s). Do not inject the oil directly into the upper channel hole (see Fig. 5.2). 10. Place the gasket on top of the cartridge and fix using the pins and T-hooks (Fig. 5.6). Fig. 5.6. Attach white rubber gasket to the dMDA cartridge. First insert pins and then T-hooks.
2. When the “Open” or “Next” button has been pressed, the screen shifts to “Please insert/remove cartridge” and “Close”. 3. Place the loaded cartridge with the holder in the Xdrop™ instrument drawer. Make sure that the cartridge is correctly positioned into the drawer by aligning the rounded corner on the holder to the rounded corner on the instrument drawer (Fig. 5.8). Incorrect insertion of the cartridge may cause damage to the instrument.
Fig. 5.9. The Xdrop™ instrument “Select cartridge type” screen. Select dMDA cartridge. 6. The channels to be processed are selected by pressing the corresponding numbers 1-8 on the screen. When selected, the button turns green (green = selected & blue = not selected) (Fig. 5.10). Deselect the channels not used. Fig. 5.10. Selecting the channels to be used. Selected channels will be indicated by green buttons (in this figure, channels 1 to 4). Blue buttons indicate channels not yet selected.
7. Press “run”. The message “Making your droplets” and the remaining run time is displayed on the screen (Fig.5.11). The dMDA protocol will produce droplets in approximately 45 seconds. Fig. 5.11. Touch screen image while producing droplets. 8. Once droplet production has been completed, the screen will change to “Your droplets are ready”. 9. Press “Open” to eject the cartridge. 10. Remove the cartridge from the instrument and place it in a LAF hood. 11.
15. Inspect the volume of collected droplets before removing the oil (step 16). You can expect approximately 2-3 mm layer of droplets on top of the oil phase. 16. Remove the excess dMDA oil from the bottom of the collection PCR tube. Only 1-2 mm of dMDA oil should be left in the bottom of the tube (see Fig. 5.12). dMDA droplets (2-3 mm) Oil (1-2 mm) Fig. 5.12. Collection of dMDA droplets from the collection well. Left: Drawing of collection well.
Fig. 5.13. Place a transparent protective film on the dMDA cartridge to seal the wells to avoid crosscontamination.
Chapter 6: Evaluation of Amplification and Target Enrichment Quantify total DNA After dMDA incubation, break the dMDA droplets with Break solution ● and Break colour ● (Fig. 6.1). 1. Add 20 μl Break solution ● to each tube. 2. Add 1 μl of Break colour ●. This will colour the water phase. If colouring is too weak, add 1 extra μl. Note: The water phase may be a colour ranging from yellow to purple as the Break colour is functioning as a pH indicator as well. 3. Flick tube gently, do not vortex. 4.
Evaluate the enrichment of target DNA After quantification of total DNA continue with measurement of target enrichment. An online tool for calculating the DNA enrichment based on qPCR is available at samplix.com To determine fold enrichment of target DNA, perform a qPCR using the qPCR QC primers, not overlapping with the dPCR enrichment amplicon(s). See “Droplet PCR Reaction Design for Target Enrichment” (Chapter 2) or the online primer design tool at samplix.com.
Fig. 6.2 Example of amplification and standard curves for qPCR QC validation of enrichment. Left: In the Amplification plot, the curve in red represents the dMDA amplified sorted positive population, while the grey shades represent 10x, 1x and 0,1x reference DNA (from darker to lighter). Right: Standard curve and calculations of PCR efficiency. Calculate fold enrichment of target DNA Use the “Enrichment calculator” in the “Tool” section under “Resources” on the Samplix homepage (Fig. 6.
Example: Calculate Enrichment based on Targets per genome If you select the tool “Calculate Enrichment based on Targets per genome”, the following information is required: • • Genome size: Size of (host) genome in base pairs. PCR efficiency: Efficiency of PCR reaction in %. You can use the efficiency calculated when validating primers designed (if same qPCR reagents have been used). Calculate the PCR efficiency using the Ct values as input with the formula: (10^(-1/slope)-1) *100.
The calculator will provide you with the assessment of a successful enrichment of your DNA sample, as well as the estimated concentration of target after enrichment. After enrichment verification, you can proceed to prepare your DNA libraries for the desired sequencing platform. XdropTM enriched DNA is compatible with both long- and short-read sequencing technology. Please contact us at support@samplix.com for library preparation and sequencing recommendations.
