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Protocols
MiniSeq

MiniSeq

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

If you plan to sequence on the same day, place the flow cell at room temperature and put the reagent cartridge in a water bath to thaw before starting your pool. The flow cell needs to equilibrate for at least half an hour and the reagent cartridge needs even more time to thaw. You can also thaw the reagent cartridge in the fridge or cold room overnight. Once thawed, use within a week.

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 enter into the calculator sheet to check that your pool is close to the nM concentration you normalized to.

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 pooled libraries, 20pM denatured PhiX, 1M Tris-HCl ph 8, 1N NaOH, MilliQ water, RSB, and hybridization buffer.

  1. In a 1.5mL tube labeled "1 nM", add the following, according to pool nM concentration:

1nM dilution

Library Pool nM Concentrationul RSBul Library Pool
10
90
10
4
75
25
2
50
50

2. 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.

3. In another 1.5mL tube, make a 200mM dilution of Tris by adding 800uL of MilliQ water and 200uL 1M Tris. Vortex and set aside.

2. In another 1.5mL tube labeled "5 pM," combine 5ul 1nM pool with 5ul 0.1 N NaOH and pipette to mix.

3. Incubate for 5 minutes at room temperatures.

7. Immediately 5ul 200mM Tris and pipette to mix.

8. Add 985ul of hybridization buffer. Slowly pipette to mix or invert the tube. Your "5pM" tube now contains your denatured library pool at 5pM concentration.

10. 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 PhiX:

MiniSeq Final Dilution

Desired Loading Concentration pMul Hyb Bufferul 5pM Library Poolul 20pM PhiX for 1% Spike-Inul 20pM PhiX for 5% Spike-Inul 20pM PhiX 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

11. Invert to mix and keep on ice until ready to use.

DRAFT Loading the Sequencer

Before loading, make sure your run has been programmed in Local Run Manager or another sample sheet template.

1. Make sure the flow cell has been at room temperature for at least 30 minutes to equilibrate. Bring it to the sequencer, open the container, and remove the flow cell.

2. Hold the flow cell to the light and inspect the for any scratches or defects.

3. Put some ethanol 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.

4. Press "Sequence" and log in to Basespace.

5. Following the on-screen prompts, load the flow cell and select Next. The pins on the machine line up with the holes on the flow cell. Press the white button to release the flow cell and push the clamp down gently to secure it.

6. Remove the Spent Reagents Container. There is a chemical waste carboy around the corner from the sequencer. The container does not have a lid. Empty it 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.

7. 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.

8. Return to your new, thawed reagent cartridge. Gently tilt it back and forth to check thaw and mix reagents.

9. Hold a p1000 tip in your hand and stab into the foil of well #10 on the reagent cartridge. Open up the foil of the well completely.

10. Pipette your denatured, diluted pool into the well. Pipette the entire contents of the well, including any that got into the cap. You will have about ~1300ul total.

11. Load the reagent cartridge into the sequencer.

12. Follow the on-screen prompts, selecting your Basespace run and confirming the parameters.

13. After the self-check, press Start!