TITLE: C-SOP-601: Operation of the Illumina MiSeq for Whole Genome Sequencing (WGS)
AUTHORS: Mihir Kekre, Ben Pascoe

[DESCRIPTION]
The Illumina® MiSeq™ system combines proven sequencing by synthesis (SBS) technology with a revolutionary workflow that lets you go from DNA to analysed data in as few as 48 hours. The MiSeq integrates cluster generation, sequencing, and data analysis on a single instrument.

This method describes the operation and maintenance of a MiSeq instrument used to perform whole-genome sequencing on human and non-human (bacterial, viral etc.) short-fragment libraries and the preliminary run performance analysis of the data generated as a result.

This protocol has been adapted from the technology and methods developed by Illumina Inc.


This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
 

[GUIDELINES]
Storage:
1. MiSeq Reagent Kit:
Box 1 of 2: 
Store at -15°C to -25°C, away from light 
MiSeq Cartridge 
Hybridisation Buffer (HT1) 

Box 2 of 2:
Store at 2-8°C 
Incorporation Buffer (PR2) 
Flow Cell

2. Remaining materials: 
Refer to manufacturer's instructions.

[STEPS]
SECTION: Before Starting
1. Prior to initiating the protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared according to local GLP guidelines for molecular methods.

Create an organised bench space by clearing away all clutter in order to maximize work efficiency and to avoid unnecessary movements that will minimise exposure of sterile materials to airborne and liquid contaminants. 

Prepare a large bucket of ice to store reagents and samples temporarily during use.
SECTION: Instrument Initialisation
6. Power on the instrument using the power switch located on the back of the instrument.
SECTION: Instrument Initialisation
7. If the MiSeq is networked, enter your Illumina BaseSpace account username and password. If you do not have an account, sign up to register for one here.
SECTION: Instrument Initialisation
8. After the MiSeq computer has booted up, the MiSeq Control Software (MCS) will load automatically. Wait for the machine to complete initialisation.
SECTION: Library Denaturation and Dilution (Standard Method)
9. Follow the steps most appropriate for your library and the version of MiSeq reagent kit that is in use (as detailed in Table 2)

For the NexteraTM DNA Flex Library Prep Kit, see dilute and denature directions in the Nextera DNA Flex Library Prep Reference Guide (document # 1000000025416).   

SECTION: Before Starting
3. Prepare a fresh aliquot of sodium hydroxide (NaOH) by combining the reagents below:
200 µL of 1.0 N NaOH
800 µL of Nuclease-free water
The result is a 1 mL of 0.2N NaOH.
 


SECTION: Before Starting
4. Remove the HT1 buffer from the deep freezer (-20 °C) storage and thaw it at room temperature. 

Store at 4 °C until ready for use.
 


SECTION: Library Denaturation and Dilution (Standard Method)
10. Combine the following reagents in a microcentrifuge tube:
5 µL of the 4nM library sample or pool.
5 µL of 0.2N NaOH

SECTION: Library Denaturation and Dilution (Standard Method)
11. Vortex for 10 s and centrifuge in a tabletop tube spinner for1 min.

SECTION: Library Denaturation and Dilution (Standard Method)
12. Incubate on the bench at Room temperature for 5 min.

SECTION: Library Denaturation and Dilution (Standard Method)
13. Add 990 µL of chilled HT1 buffer to the tube containing the denatured library sample.

The resulting solution will now be 1 mL of a 20pM denatured library.

SECTION: Library Denaturation and Dilution (Standard Method)
14. Dilute the 20pM library to the desired concentration using the volumes in Table 3a below:
  
Invert several times to mix and pulse centrifuge the solution.

This is now your library loading concentration.
 


SECTION: Library Denaturation and Dilution (Standard Method)
10. Combine the following reagents in a microcentrifuge tube:
5 µL of the 2nM library sample or pool.
5 µL of 0.2N NaOH
SECTION: Library Denaturation and Dilution (Standard Method)
11. Vortex for 10 s and centrifuge in a tabletop tube spinner for1 min.
SECTION: Library Denaturation and Dilution (Standard Method)
12. Incubate on the bench at Room temperature for 5 min.
SECTION: Library Denaturation and Dilution (Standard Method)
13. Add 990 µL of chilled HT1 buffer to the tube containing the denatured library sample.

The resulting solution will now be 1 mL of a 10pM denatured library.
SECTION: Library Denaturation and Dilution (Standard Method)
14. Dilute the 10pM library to the desired concentration using the volumes in Table 3b below:

  
Invert several times to mix and pulse centrifuge the solution.

This is now your library loading concentration.
 

SECTION: PhiX (Control) Denaturation and Dilution
15. To spike PhiX as a sequencing performance control into the run, use Table 4 and the subsequent steps to dilute and denature the PhiX stock.
  

SECTION: PhiX (Control) Denaturation and Dilution
16. Combine the following reagents in a microcentrifuge tube:
2 µL of the 10nM PhiX library.
3 µL of 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20
 

The resulting solution is a 4nM PhiX library.
SECTION: PhiX (Control) Denaturation and Dilution
17. Combine the following reagents in a microcentrifuge tube:
5 µL of the 4nM PhiX library.
5 µL of 0.2N NaOH
SECTION: PhiX (Control) Denaturation and Dilution
18. Vortex for 10 s and centrifuge in a tabletop tube spinner for1 min.
SECTION: PhiX (Control) Denaturation and Dilution
19. Incubate on the bench at Room temperature for 5 min.
SECTION: PhiX (Control) Denaturation and Dilution
20. Add 990 µL of chilled HT1 buffer to the tube containing the denatured library sample.

The resulting solution will now be 1 mL of a 20pM denatured PhiX library.

Invert several times to mix.
SECTION: PhiX (Control) Denaturation and Dilution
21. If using a MiSeq Reagent v3 kit, no further dilution is required.
If using a MiSeq Reagent v2,

Combine the following in a 2.0 mL microcentrifuge tube:
375 µL of the 20pM denatured PhiX library.
225 µL of chilled buffer HT1.

The resulting solution is 600 µL of a 12.5pM denatured PhiX library.

Invert several times to mix.


SECTION: PhiX (Control) Denaturation and Dilution
22. For most libraries, a low concentration of PhiX control spike-in of 1% is adequate as a sequencing control. For low diversity libraries, increase the PhiX control spike-in to at least 5%.

Combine the following volumes from Table 5 to obtain your final loading spike-in of PhiX for the run.
  

Set aside on ice until it is ready to load into the reagent cartridge.
SECTION: Before Starting
5. For best results, step 5 and its sub-steps should be performed 2 hours prior to sample loading and run set up.

Allow adequate time for thawing of the reagent cartridge. Table 1 details the conditions needed to ensure optimal defrosting.
 


SECTION: Before Starting
5.1. Remove the reagent cartridge from the -20 °C storage.

SECTION: Before Starting
5.2. Place the reagent cartridge in a water bath containing enough room temperature deionized/MilliQ water to submerge the base of the cartridge. 

Do NOT allow the water to exceed the maximum water line printed on the cartridge (as shown below).

  

SECTION: Before Starting
5.3. Allow the reagent cartridge to completely thaw in the room temperature water bath. 
MiSeq v3 cartridges— ~ 90 min. 
MiSeq v2 cartridges— ~ 60 min.

SECTION: Before Starting
5.4. Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. 

Dry the base of the cartridge.
SECTION: Before Starting
5.5. Invert the reagent cartridge ten times to mix the thawed reagents, and then inspect that all positions are thawed.
SECTION: Before Starting
5.6. Inspect the reagents in positions 1, 2, and 4 to make sure that they are fully mixed and free of precipitates or icy blocks.
SECTION: Before Starting
5.7. Gently tap the cartridge on the bench to reduce air bubbles in the reagents.
 

SECTION: Before Starting
5.8. Set aside the thawed reagent cartridge at 4 °C until the run is ready for setup. 

SECTION: Setting Up a Run on the MiSeq
23. Prior to setting up the run, ensure that there is enough free disk space (100 GB) on the instrument. 
 

SECTION: Preparing and Loading the Flow Cell
26. Retrieve the Flow Cell and Incorporation buffer (PR2) stored at 4 °C.

SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
35. Open the reagent compartment door and raise the sipper handle until it locks into place.
SECTION: Before Starting
2. An overview of the MiSeq instrument, software and reagent kit components can be found on pg. 3-11 of the MiSeq Systems Guide.
SECTION: Loading Sample Library onto the Cartridge
41. Remove the thawed reagent cartridge from 4°C storage (or water bath), ensuring that it has been thoroughly dried and the reagents mixed by gentle inversion.
SECTION: Setting Up a Run on the MiSeq
24. Ensure that the Sample Sheet (and Sample Plate, if applicable) setup has been completed using Illumina Experiment Manager (IEM) on the instrument.
SECTION: Setting Up a Run on the MiSeq
25. Open the MiSeq Control Software window, select “Sequence” and proceed immediately to Preparing and Loading the Flow Cell.
 

SECTION: Setting Up a Run on the MiSeq
23.1. BaseSpace Users: It is recommended to maintain a duplicate copy of the run folder locally on the instrument as a backup. Doing the following will ensure that a run folder will be generated in the Data drive file folders, in addition to being streamed to BaseSpace. Follow the procedure below:
SECTION: Setting Up a Run on the MiSeq
23.2. From the MCS home screen, select “Run Options.”
SECTION: Setting Up a Run on the MiSeq
23.3.  Under the “Run Settings” tab, check the “When using BaseSpace, replicate analysis locally on MiSeq” checkbox.
SECTION: Setting Up a Run on the MiSeq
23.4. Select “Save and Return” to save these changes and return to the home screen.
SECTION: Loading the Reagent Cartridge onto the Instrument
45. On the instrument screen, confirm it says “Load Reagent Cartridge”.

This will ensure the sippers in the chiller have been raised to avoid snapping them when removing the wash tray.
SECTION: Preparing and Loading the Flow Cell
27. Wearing clean powder-free gloves, and using plastic tweezers, grip the flow cell by the base of the plastic cartridge and remove it from the flow cell container (as shown below).
   

SECTION: Preparing and Loading the Flow Cell
28. Lightly rinse the flow cell with laboratory-grade water until both the glass and plastic cartridge are thoroughly rinsed of excess salts.
  
 


SECTION: Preparing and Loading the Flow Cell
29. Thoroughly dry the flow cell and cartridge with a lint-free lens cleaning tissue. Gently pat dry in the area of the back port gasket and adjacent glass.
  
 

  

SECTION: Preparing and Loading the Flow Cell
30. Dry excess alcohol with a lint-free lens cleaning tissue.

Make sure that the flow cell ports are free of obstructions and that the gasket is well-seated around the flow cell ports.

If the gasket appears to be dislodged, gently press it back into place until it sits securely around the flow cell ports.
SECTION: Preparing and Loading the Flow Cell
31. Raise the flow cell compartment door, and then press the release button to the right of the flow cell clamp (as shown below).

The flow cell clamp opens.
  
 

SECTION: Preparing and Loading the Flow Cell
32. Holding the flow cell by the edges, place it on the flow cell stage (as shown below).
  

SECTION: Preparing and Loading the Flow Cell
33. Gently press down on the flow cell clamp to close it over the flow cell. 

As the flow cell clamp closes, alignment pins position the flow cell. An audible click indicates that the flow cell clamp is secure.
  

SECTION: Preparing and Loading the Flow Cell
34. The software will now identify and indicate the Flow Cell's RFID tag to confirm successful placement. 

Close the Flow Cell compartment door and hit Next.
 

SECTION: Loading Sample Library onto the Cartridge
42. Tap the reagent cartridge on a flat hard surface to gather the contents at the bottom of the reservoirs.
SECTION: Loading Sample Library onto the Cartridge
43. Using a clean 1000 µl pipette tip, pierce the foil seal over the reservoir labeled “Load Sample.”
SECTION: Loading Sample Library onto the Cartridge
44. Pipette 600 µL of the denatured DNA library into the “Load Sample” reservoir (as shown below).

Tap gently on a hard surface to gather all the contents to the bottom.
 

  

SECTION: Loading the Reagent Cartridge onto the Instrument
46. Open the reagent chiller door, remove the wash tray, and dry bottom of chiller compartment if necessary using absorbent wipes. 
 

SECTION: Loading the Reagent Cartridge onto the Instrument
47. Hold the reagent cartridge on the end with the Illumina label, and slide the reagent cartridge into the reagent chiller until the cartridge stops (as shown below).
  
 


SECTION: Loading the Reagent Cartridge onto the Instrument
48. Close the chiller door and confirm that the RFID of the reagent cartridge has been read successfully.
 

Also close the reagent compartment door.
SECTION: Setting Up the Sample Sheet
49. Every run must have a sample sheet. By default, the software looks for a sample sheet file with a name matching the barcode number of the reagent cartridge loaded on the instrument. If a sample sheet is not found, a message appears that prompts you to browse to the location of the correct sample sheet for your run.

On the subsequent screen, select Change Sample Sheet and then select Browse to navigate to the appropriate sample sheet.
 

SECTION: Setting Up the Sample Sheet
50. Select the desired sample sheet and click OK.

Hit Save and Continue, and then Next. Your sample sheet is now successfully loaded.
 

SECTION: Starting the Run
51. After loading the flow cell and reagents, review the run parameters and perform a pre-run check before starting the run.
SECTION: Starting the Run
52. Review Run Parameters:

1. Review Experiment Name, Analysis Workflow, and Read Length. These parameters are specified in the sample sheet.
 


2. Select Change Folder to review the folder locations. 
 


3. Modify 1. and 2. as needed and then select Save, and then Next.
SECTION: Starting the Run
53. Review Pre-Run Check:

The system performs a check of all run components, disk space, and network connections before starting the run.

When all items successfully pass the pre-run check, select Start Run.
 

SECTION: Post-Run Wash and Reagent Disposal
54. The post-run wash is the standard instrument wash performed between sequencing runs. Always perform an instrument wash after completing a run. 
Regular instrument washes ensure continued performance in the following ways: 
Flushes any remaining reagents from the fluidics lines and sippers 
Prevents salt accumulation and crystallisation in the fluidics lines and sippers 
Prevents cross-contamination from the previous run 

Follow the on-screen prompts to load the wash components and perform the wash. 

The post-run wash takes approximately 20 min.

If you are using MCS v2.5 or later, you have the option to perform a post-run wash that includes a template line wash with sodium hypochlorite solution (NaOCl). The wash takes approximately 30 min. 

Maintenance and Standby washes are detailed on pg. 34 and 36 of the MiSeq System Guide.
 


SECTION: Monitoring Run Performance
64. During and after the run, monitor run progress, intensities, and quality scores that appear on the Sequencing screen. 

Alternatively, if you are connected to BaseSpace, the run can be monitored real-time using SAV in BaseSpace or using the BaseSpace Hub on mobile. 
Run Progress—Shows run progress in a status bar and lists the number of cycles completed. 
Intensity—Shows the value of cluster intensities of the 90th percentile for each tile.
 

 

SECTION: Exporting Data from the MiSeq
65. Data is found on the following path:
D:\ → Illumina → MiSeq Output → Run folder (choose the most recent run folder) → Data → Intensities → BaseCalls. 

Within the BaseCalls folder, the raw read data is in a fastq.gz format. 

There will be 2 files per isolate for paired reads (R1 and R2). This data can be copied onto an external hard drive and moved to a computer workstation. 

Alternatively, the MiSeq instrument can also be connected to a local network share drive to directly transfer files from the instrument.
 

SECTION: Post-Run Wash and Reagent Disposal
55. While the MiSeq run is underway:

1. Prepare a fresh wash solution of Tween 20 with nuclease-free water:
a. Add 5 mL 100% Tween 20 to 45 mL nuclease-free water. This results in a 10% Tween 20. 

b. Add 25 mL 10% Tween 20 to 475 mL nuclease-free water. This results in a 0.5% Tween 20 wash solution. 

c. Invert five times to mix.



2. Prepare fresh sodium hypochlorite (NaOCl) wash solution with nuclease-free water:
a. Add 36 µL of 5% NaOCl to 864 µL nuclease-free water. This results in a 1:25 NaOCl dilution. 

b. Add 50 µL of the 1:25 NaOCl dilution to 950 µL of nuclease-free water in a MiSeq Disposable Wash Tube.

 

3. Prepare the wash components with fresh wash solution: 
a. Add 6 mL wash solution to each reservoir of the wash tray. 

b. Add 350 mL wash solution to the 500 ml wash bottle.


 

SECTION: Instrument Shutdown &amp; Reboot
66. The instrument should be rebooted weekly and after a file deletion to free any virtual memory. 

On the MCS home screen, select Manage Instrument then push “Reboot”. It will take approximately 10 min for the system to reboot and restart the MiSeq Control software.
SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
36. Remove the bottle of PR2 from 4 °C storage. Invert several times to mix, and then remove the lid.

SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
37. Remove the wash bottle and load the PR2 bottle.  

SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
38. The software will now identify and indicate the RFID tag of the PR2 bottle to confirm successful placement. 

Close the reagent compartment door and hit Next.
 

SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
39. Empty the contents of the waste bottle into the appropriate waste container.

Reload the empty bottle back into its appropriate slot in the compartment.
SECTION: Incorporation Buffer (PR2) and Waste Bottle Check
40. Slowly lower the sipper handle ensuring that the sippers lower into the PR2 and waste bottles.  

SECTION: Post-Run Wash and Reagent Disposal
57. Once the run is complete, select the “Start Wash” option at the bottom right of the screen. The software automatically raises the sippers in the reagent chiller.
 

SECTION: Post-Run Wash and Reagent Disposal
56. Insert the MiSeq Wash Tube containing 0.01% NaOCl wash solution into position 17 of the MiSeq wash tray.

The tube displaces the Tween 20 and laboratory-grade water wash solution from position 17.
  
 


SECTION: Post-Run Wash and Reagent Disposal
58. Open the reagent compartment door and reagent chiller door, and remove the used reagent cartridge from the chiller.
SECTION: Post-Run Wash and Reagent Disposal
59. Slide in the wash tray into the reagent chiller until it stops, and then close the reagent chiller door.
SECTION: Post-Run Wash and Reagent Disposal
60. Raise the sipper handle in front of the PR2 bottle and waste bottle until it locks into place.

Remove the PR2 bottle and replace it with the wash bottle.
 

SECTION: Post-Run Wash and Reagent Disposal
61. Remove the waste bottle and discard the contents appropriately. Return the waste bottle to the reagent compartment.
SECTION: Post-Run Wash and Reagent Disposal
62. Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.
SECTION: Post-Run Wash and Reagent Disposal
63. Close the reagent compartment door.

Select Next.
 


SECTION: Instrument Maintenance
68. The following maintenance protocols are to be carried out at the recommended intervals.


 

 

SECTION: Instrument Shutdown &amp; Reboot
67. Alternatively, the instrument can be power cycled by selecting Manage Instrument on the home screen and then “Shut Down”. 

Once the instrument has shut down (noted by a distinct click sound), reach behind the right-hand side of the instrument and flip the power switch located near the power cord. 

Let the instrument power down for at least 10 min and then flip the switch back on. The instrument will boot up the MCS as normal.
SECTION: Additional Information and Troubleshooting
69. Illumina MiSeq Workflow Overview

MiSeq System Denature and Dilute Libraries Guide (15039740)

MiSeq System Guide (1000000061014)
"Troubleshooting Appendix" - pg. 41 onwards

MiSeq Sample Sheet: Quick Reference Guide (15028392)
"Creating a New MiSeq Sample Sheet" (pg. 17-18)

MiSeq Performance Specifications

Sequence Analysis Viewer (SAV) Software User Guide (15020619)

MiSeq Reporter (MSR) Software Guide (15042295)
"Re-queuing a Run for Analysis" (pg. 19-20)

Illumina Experiment Manager (IEM) Software Guide (15031335)

"The five Quality metrics every NGS user should know", Perkin-Elmer Horizon Discovery.