CHMI owns and operates its own MiniSeq. This instrument is ideal for a bacterial genome sequencing, PCR panel assays, and low-coverage QC runs prior to larger NovaSeq runs.
- Reference material
- Pooling
- Thawing the Kit
- Denaturation and Dilution
- Local Run Manager
- Loading the Sequencer
Reference material
- Sequencing by Synthesis: 5 minute YouTube video
- MiniSeq system guide
- MiniSeq denature and dilute guide
- MiniSeq support site
- MiniSeq training videos
Pooling
Quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library.
Run your samples on Tapestation with either the D1000, HSD1000, or HSD5000 assay, depending on library prep kit. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
Download our nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. Normalize and pool all your libraries to 10, 4, 2 nM in a LoBind microcentrifuge tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
Quantify your pool on Qubit and Tapestation.
Enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.
Thawing the Kit
Thaw the kit in one of two ways:
Thaw the reagent cartridge in the fridge overnight
Thaw the reagent cartridge in a water bath for 90 minutes if sequencing that day
Once thawed, the kit must be used within a week.
Place the flow cell at room temperature for 30 minutes before use.
Thaw Hybridization Buffer on ice for 30 minutes.
Denaturation and Dilution
The following protocol is based on the MiniSeq Denature and Dilute Guide (linked above), with few adjustments. The biggest change is that we do not vortex the libraries after denaturation.
Before proceeding to these steps, make sure your flow cell is out at room temperature and your reagent cartridge is thawing. In addition to these sequencing reagents, you will need your pool, 20pM denatured PhiX, 1M Tris-HCl pH 8, 1N NaOH, MilliQ water, RSB, and thawed Hybridization Buffer.
In a 1.5mL tube labeled "1 nM", add the following, according to pool nM concentration:
In a 1.5mL tube, make a 0.1N dilution of NaOH by adding 900uL of MilliQ water and 100uL 1N NaOH. Vortex and set aside.
In another 1.5mL tube, make a 200mM dilution of Tris-HCl by adding 800uL of MilliQ water and 200uL 1M Tris-HCl. Vortex and set aside.
In another 1.5mL tube labeled "5 pM," combine 5 ul 1nM pool with 5 ul 0.1 N NaOH and pipette to mix.
Incubate for 5 minutes at room temperatures.
After the 5 minutes are up, immediately 5 ul 200mM Tris-HCl and pipette to mix.
Add 985 ul of Hybridization Buffer. Slowly pipette to mix or invert the tube. Your "5pM" tube now contains your denatured library pool at 5pM concentration.
Proceed to the final dilution of your samples. Label your new tube "Final" and add the appropriate volumes of Hybridization Buffer, 5pM library, and 20pM denatured PhiX:
[Desired Loading] pM | ul Hyb Buffer | ul 5 pM Library Pool | ul 20 pM PhiX for 1% Spike-In | ul 20 pM PhiX for 5% Spike-In | ul 20pM Phi X for 20% Spike-In |
|---|---|---|---|---|---|
1.3 | 370 | 130 | 0.325 | 1.63 | 6.5 |
1.4 | 360 | 140 | 0.350 | 1.75 | 7.0 |
1.5 | 350 | 150 | 0.375 | 1.88 | 7.5 |
1.6 | 340 | 160 | 0.400 | 2.00 | 8.0 |
1.7 | 330 | 170 | 0.425 | 2.13 | 8.5 |
1.8 | 320 | 180 | 0.450 | 2.25 | 9.0 |
Invert to mix and keep on ice until ready to use.
Local Run Manager
At the sequencer, minimize the sequencing software
Select the icon for Local Run Manager (looks like a blue version of the Google Chrome icon)
Click the button to start a new run
Input the following
Run name
Run description
Sample names
Index sequences (or index wells)
Read lengths
Save and finish
Loading the Sequencer
Before loading, make sure your run has been programmed in Local Run Manager (on the MiniSeq) or another sample sheet template.
Make sure the flow cell has been at room temperature for at least 30 minutes to equilibrate. Bring it to the sequencer.
Press "Sequence" and log in to Basespace.
Open the flow cell container and remove the flow cell. Hold the flow cell to the light and inspect the for any scratches or defects.
Put some alcohol on a kim wipe, and slowly wipe the glass face of the flow cell in a single direction. Take a clean kim wipe and repeat to remove any excess ethanol or remaining dust.
Inspect the flow cell ports for obstruction.
Load the flow cell. Press the white button to release the old flow cell and push the clamp down gently to secure the new flow cell. The pins on the machine line up with the holes on the flow cell. Select Next.
Remove the Spent Reagents Container. The container does not have a lid. There is a chemical waste carboy in our lab under the themocyclers. Empty any spent reagents into the waste carboy near the NextSeq and return to its position. This waste contains formamide. Never dispose it down the sink and change gloves if you spill or drip.
Remove the old Reagent Cartridge from the sequencer. Remove the reservoir behind position #9. This contains trace amounts of formamide and is disposed in a bag for chemical waste pickup. The rest of the cartridge can be disposed in regular trash.
Return to your new, thawed reagent cartridge. Gently tilt it back and forth to check thaw.
Invert the cartridge 5 times to mix reagents. Gently tap the cartridge on the benchtop to reduce air bubbles.
Hold a p1000 tip in your hand and stab into the foil of well #16 on the reagent cartridge. Open up the foil of the well completely.
Pipette the entire 500 µL your denatured, diluted pool into well #16. Remember to grab all liquid, including any that got into the cap.
Load the reagent cartridge into the sequencer.
Follow the on-screen prompts, select your Basespace run, and confirm the parameters.
After the self-check, press Start!