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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block">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.</div><div class = "text-block">This protocol has been adapted from the technology and methods developed by Illumina Inc.</div><div class = "text-block"><span>This is an open-access protocol distributed under the terms of the </span><a href="https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Creative Commons Attribution-NonCommercial-ShareAlike</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/files/cuzrbf8zp.png?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWSNCI5SNCPTWTQQ%2F20210112%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20210112T173149Z&X-Amz-Expires=604800&X-Amz-SignedHeaders=host&X-Amz-Signature=1b2f1a454c966fddb1af93dd8213eb2cd4cbeb804c6ceee4a5e1185f39d2f243" /></div></div>

[STEPS]
?. [Before Starting]
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.
?. [Instrument Initialisation]
Power on the instrument using the power switch located on the back of the instrument.
?. [Instrument Initialisation]
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.
?. [Instrument Initialisation]
After the MiSeq computer has booted up, the MiSeq Control Software (MCS) will load automatically. Wait for the machine to complete initialisation.
?. [Library Denaturation and Dilution (Standard Method)]
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).
?. [Before Starting]
Prepare a fresh aliquot of sodium hydroxide (NaOH) by combining the reagents below: of 1.0 N NaOH of Nuclease-free waterThe result is a  of 0.2N NaOH.
200 µl
800 µl
1 mL
Use the fresh dilution within 8 hours of preparation.
?. [Before Starting]
Remove the HT1 buffer from the deep freezer () storage and thaw it at room temperature. Store at  until ready for use.
-20 °C
4 °C
Buffer HT1 has a high salt concentration and can take long periods to thaw out completely. Defrost this reagent sufficiently ahead of the run.
?. [Library Denaturation and Dilution (Standard Method)]
Combine the following reagents in a microcentrifuge tube: of the 4nM library sample or pool. of 0.2N NaOH
5 µl
5 µl
?. [Library Denaturation and Dilution (Standard Method)]
Vortex for  and centrifuge in a tabletop tube spinner for.
?. [Library Denaturation and Dilution (Standard Method)]
Incubate on the bench at  for .
0 Room temperature
?. [Library Denaturation and Dilution (Standard Method)]
Add  of chilled HT1 buffer to the tube containing the denatured library sample.The resulting solution will now be  of a 20pM denatured library.
990 µl
1 mL
?. [Library Denaturation and Dilution (Standard Method)]
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.
For bacterial WGS at 300-cycle chemistry, we recommend diluting the denatured 20pM library down to either 10pM depending on the cluster metrics required.
?. [Library Denaturation and Dilution (Standard Method)]
Combine the following reagents in a microcentrifuge tube: of the 2nM library sample or pool. of 0.2N NaOH
5 µl
5 µl
?. [Library Denaturation and Dilution (Standard Method)]
Vortex for  and centrifuge in a tabletop tube spinner for.
?. [Library Denaturation and Dilution (Standard Method)]
Incubate on the bench at  for .
0 Room temperature
?. [Library Denaturation and Dilution (Standard Method)]
Add  of chilled HT1 buffer to the tube containing the denatured library sample.The resulting solution will now be  of a 10pM denatured library.
990 µl
1 mL
?. [Library Denaturation and Dilution (Standard Method)]
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.
For bacterial WGS at 300-cycle chemistry, we recommend diluting the denatured 20pM library down to either 10pM depending on the cluster metrics required.
?. [PhiX (Control) Denaturation and Dilution]
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.
?. [PhiX (Control) Denaturation and Dilution]
Combine the following reagents in a microcentrifuge tube: of the 10nM PhiX library. of 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20The resulting solution is a 4nM PhiX library.
2 µl
3 µl
Always prepare a fresh dilution of PhiX and use within 12 hours.
?. [PhiX (Control) Denaturation and Dilution]
Combine the following reagents in a microcentrifuge tube: of the 4nM PhiX library. of 0.2N NaOH
5 µl
5 µl
?. [PhiX (Control) Denaturation and Dilution]
Vortex for  and centrifuge in a tabletop tube spinner for.
?. [PhiX (Control) Denaturation and Dilution]
Incubate on the bench at  for .
0 Room temperature
?. [PhiX (Control) Denaturation and Dilution]
Add  of chilled HT1 buffer to the tube containing the denatured library sample.The resulting solution will now be  of a 20pM denatured PhiX library.Invert several times to mix.
990 µl
1 mL
?. [PhiX (Control) Denaturation and Dilution]
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: of the 20pM denatured PhiX library. of chilled buffer HT1.The resulting solution is  of a 12.5pM denatured PhiX library.Invert several times to mix.
375 µl
225 µl
600 µl
?. [PhiX (Control) Denaturation and Dilution]
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.
?. [Before Starting]
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.Method of thawingThawing timeShelf-life of thawed cartridge (at 4ºC or on ice)Ambient temperature water bath (opened)1.5 hoursup to 24 hours2-8ºC (sealed)12 hoursup to 4 days
Method of thawingThawing timeShelf-life of thawed cartridge (at 4ºC or on ice)Ambient temperature water bath (opened)1.5 hoursup to 24 hours2-8ºC (sealed)12 hoursup to 4 days
?. [Before Starting]
Remove the reagent cartridge from the  storage.
-20 °C
?. [Before Starting]
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).
?. [Before Starting]
Allow the reagent cartridge to completely thaw in the room temperature water bath. MiSeq v3 cartridges— ~ . MiSeq v2 cartridges— ~ .
?. [Before Starting]
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.
?. [Before Starting]
Invert the reagent cartridge ten times to mix the thawed reagents, and then inspect that all positions are thawed.
?. [Before Starting]
Inspect the reagents in positions 1, 2, and 4 to make sure that they are fully mixed and free of precipitates or icy blocks.
?. [Before Starting]
Gently tap the cartridge on the bench to reduce air bubbles in the reagents.
The MiSeq's sipper tubes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bubbles.
?. [Before Starting]
Set aside the thawed reagent cartridge at  until the run is ready for setup.
4 °C
?. [Setting Up a Run on the MiSeq]
Prior to setting up the run, ensure that there is enough free disk space (100 GB) on the instrument.
If there is less than 100 GB is available, check the Instrument Maintenance section of the MiSeq Systems Guide.
?. [Preparing and Loading the Flow Cell]
Retrieve the Flow Cell and Incorporation buffer (PR2) stored at .
4 °C
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
Open the reagent compartment door and raise the sipper handle until it locks into place.
?. [Before Starting]
An overview of the MiSeq instrument, software and reagent kit components can be found on pg. 3-11 of the MiSeq Systems Guide.
?. [Loading Sample Library onto the Cartridge]
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.
?. [Setting Up a Run on the MiSeq]
Ensure that the Sample Sheet (and Sample Plate, if applicable) setup has been completed using Illumina Experiment Manager (IEM) on the instrument.
?. [Setting Up a Run on the MiSeq]
Open the MiSeq Control Software window, select “Sequence” and proceed immediately to Preparing and Loading the Flow Cell.
If using BaseSpace, on the following screen, check the box to ensure the user info fields are active and enter user credentials to default data streaming directly to BaseSpace.
?. [Setting Up a Run on the MiSeq]
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:
?. [Setting Up a Run on the MiSeq]
From the MCS home screen, select “Run Options.”
?. [Setting Up a Run on the MiSeq]
Under the “Run Settings” tab, check the “When using BaseSpace, replicate analysis locally on MiSeq” checkbox.
?. [Setting Up a Run on the MiSeq]
Select “Save and Return” to save these changes and return to the home screen.
?. [Loading the Reagent Cartridge onto the Instrument]
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.
?. [Preparing and Loading the Flow Cell]
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).
?. [Preparing and Loading the Flow Cell]
Lightly rinse the flow cell with laboratory-grade water until both the glass and plastic cartridge are thoroughly rinsed of excess salts.
Excess salts can affect flow cell seating on the instrument. If salts dry in the imaging area, imaging can also be affected.
?. [Preparing and Loading the Flow Cell]
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.
Wet a clean piece of lens paper with ethanol and clean the glass portion of the flowcell. The glass must be free of streaks, lint, and tissue fibres. An alcohol wipe can also be used for this step. Do not add ethanol directly to the flow cell, and avoid getting ethanol (or wipes) on the port gasket.
?. [Preparing and Loading the Flow Cell]
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.
?. [Preparing and Loading the Flow Cell]
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.
Make sure that the flow cell stage is free of lint. If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol. Carefully wipe the surface of the flow cell stage until it is clean and dry.
?. [Preparing and Loading the Flow Cell]
Holding the flow cell by the edges, place it on the flow cell stage (as shown below).
?. [Preparing and Loading the Flow Cell]
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.
?. [Preparing and Loading the Flow Cell]
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.
If the software does not identify the RFID, refer to pg. 44 of the MiSeq System Guide.
?. [Loading Sample Library onto the Cartridge]
Tap the reagent cartridge on a flat hard surface to gather the contents at the bottom of the reservoirs.
?. [Loading Sample Library onto the Cartridge]
Using a clean 1000 µl pipette tip, pierce the foil seal over the reservoir labeled “Load Sample.”
?. [Loading Sample Library onto the Cartridge]
Pipette  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.
600 µl
It is recommended that the sample be dispensed toward the bottom end of the reservoir using an extended 1000 µl pipette tip to reduce the risk of droplets forming on the sides. Avoid depositing droplets on the foil seal while the sample is loaded.
?. [Loading the Reagent Cartridge onto the Instrument]
Open the reagent chiller door, remove the wash tray, and dry bottom of chiller compartment if necessary using absorbent wipes.
Do NOT leave the reagent chiller door open for extended periods of time.
?. [Loading the Reagent Cartridge onto the Instrument]
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).
Always use the reagent cartridge associated with the type of flow cell that you loaded. If the reagent cartridge is not compatible, a message appears on the screen. Select Back to load the appropriate reagent cartridge or Home to return to the Home screen.
?. [Loading the Reagent Cartridge onto the Instrument]
Close the chiller door and confirm that the RFID of the reagent cartridge has been read successfully.Also close the reagent compartment door.
If the software does not identify the RFID, refer to pg. 44 of the MiSeq System Guide.If the reagent cartridge is not compatible with the flow cell, a message appears. Select Back to load a compatible cartridge, or select Exit to return to the Home screen.
?. [Setting Up the Sample Sheet]
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.
i. Use the Change Sample Sheet command for the following:To select a sample sheet with a name that does not match the reagent cartridge barcode number When the software prompts you to choose a different sample sheet on the Review screenii. To prevent the software from searching unsuccessfully, use the Change Sample Sheet command on the Load Reagents screen to direct the software to the appropriate sample sheet.
?. [Setting Up the Sample Sheet]
Select the desired sample sheet and click OK.Hit Save and Continue, and then Next. Your sample sheet is now successfully loaded.
Refer to the MiSeq Sample Sheet: Quick Reference Guide for details on sheet design.
?. [Starting the Run]
After loading the flow cell and reagents, review the run parameters and perform a pre-run check before starting the run.
?. [Starting the Run]
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.
If IDT Illumina Unique Dual Indexes are used, the Index Reads need to be changed to 10 cycles (instead of 8 cycles).
To change folder locations, select Change Folder and browse to a preferred location. Using this option from the Review screen changes folder locations for the current run only.
?. [Starting the Run]
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.
If any items do not pass the pre-run check, a message appears on the screen with instructions on how to correct the error. For more information, refer to pg. 44 of the MiSeq System Guide.Once the run has started, do NOT open the flow cell or reagent compartment doors, neither should the instrument monitor be engaged unless the run is to be stopped or paused.Image capture on the MiSeq is sensitive to vibration. Performing tasks that cause vibration near/on the instrument during a run could cause the run to halt or adversely impact sequencing results.
?. [Post-Run Wash and Reagent Disposal]
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 .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 . Maintenance and Standby washes are detailed on pg. 34 and 36 of the MiSeq System Guide.
Start the wash directly following the completion of a run. An instrument wash is required before you can set up a subsequent run. To perform a post-run wash at a time other than directly following a run, use the command on the Perform Wash screen to initiate the wash.Leave the used flow cell (from a previous run) on the instrument. It is mandatory that a flow cell be loaded on the instrument when performing an instrument wash.
?. [Monitoring Run Performance]
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.
The Sequencing screen is view-only. To monitor the run in greater detail, use the Sequencing Analysis Viewer (SAV) installed on a computer independent of the instrument computer. A network connection is required.For more details, refer to the Sequence Analysis Viewer (SAV) Software User Guide (15020619) and pg. 26-28 of the MiSeq System Guide.
It may take up to 1 hour for the MiSeq to generate fastq files post-run as the Real-Time Analysis Software lags behind the sequencing run.If fastq data is unavailable for a given sample(s) due to incorrect index assignment or sample sheet errors, the run can be re-queued for analysis using either the MiSeq Reporter (MSR) or BaseSpace Re-Queue tutorial
?. [Exporting Data from the MiSeq]
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.
This section is for users unable to stream to BaseSpace or in need of a secondary data repository (i.e. local server) for local data analysis or storage
?. [Post-Run Wash and Reagent Disposal]
While the MiSeq run is underway:1. Prepare a fresh wash solution of Tween 20 with nuclease-free water:a. Add  100% Tween 20 to  nuclease-free water. This results in a 10% Tween 20. b. Add  10% Tween 20 to  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  of 5% NaOCl to  nuclease-free water. This results in a 1:25 NaOCl dilution. b. Add  of the 1:25 NaOCl dilution to  of nuclease-free water in a MiSeq Disposable Wash Tube.3. Prepare the wash components with fresh wash solution: a. Add  wash solution to each reservoir of the wash tray. b. Add  wash solution to the 500 ml wash bottle.
5 mL
45 mL
25 mL
475 mL
36 µl
864 µl
50 µl
950 µl
6 mL
350 mL
Using the correct concentration of NaOCl is important. Make sure to check the percentage of NaOCl on the product label. If the concentration is too high, it can make cluster generation fail in subsequent runs. If 5% NaOCl is not available, make a 1 ml solution of 0.01% NaOCl in laboratory-grade water. Do not use NaOCl with a maintenance wash or a standby wash
?. [Instrument Shutdown & Reboot]
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  for the system to reboot and restart the MiSeq Control software.
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
Remove the bottle of PR2 from  storage. Invert several times to mix, and then remove the lid.
4 °C
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
Remove the wash bottle and load the PR2 bottle.
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
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.
If the software does not identify the RFID, refer to pg. 44 of the MiSeq System Guide.
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
Empty the contents of the waste bottle into the appropriate waste container.Reload the empty bottle back into its appropriate slot in the compartment.
?. [Incorporation Buffer (PR2) and Waste Bottle Check]
Slowly lower the sipper handle ensuring that the sippers lower into the PR2 and waste bottles.
?. [Post-Run Wash and Reagent Disposal]
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.
Tick the “Perform Optional Template Line Wash” checkbox to proceed with a Post-Run bleach wash. Bleach, or sodium hypochlorite is used to reduce carry-over of nucleic acid from previous runs.If Perform optional template line wash is not selected for the post-run wash, a message on the Run Review screen reminds you the next time you start a sequencing run.
?. [Post-Run Wash and Reagent Disposal]
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.
Ensure that the neck of the tube is flush with the tray. Make sure to insert the MiSeq tube with NaOCl into tray position 17 only. Inserting the tube in another position can make cluster generation fail in subsequent runs, and can damage the fluidic system of the MiSeq instrument.
?. [Post-Run Wash and Reagent Disposal]
Open the reagent compartment door and reagent chiller door, and remove the used reagent cartridge from the chiller.
?. [Post-Run Wash and Reagent Disposal]
Slide in the wash tray into the reagent chiller until it stops, and then close the reagent chiller door.
?. [Post-Run Wash and Reagent Disposal]
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.
Discard the PR2 bottle after each run. Do not reuse any remaining PR2 buffer.
?. [Post-Run Wash and Reagent Disposal]
Remove the waste bottle and discard the contents appropriately. Return the waste bottle to the reagent compartment.
?. [Post-Run Wash and Reagent Disposal]
Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.
?. [Post-Run Wash and Reagent Disposal]
Close the reagent compartment door.Select Next.
When the wash is complete:Leave the used flow cell, wash tray, and wash bottle containing the remaining wash solution on the instrument.The sippers remain in the down position. Leave the unused wash solution in the wash tray and wash bottle to prevent the sippers from drying out and air from entering the system.
?. [Instrument Maintenance]
The following maintenance protocols are to be carried out at the recommended intervals.Monthly Wash Schedule ScenariosWeek 1Week 2Week 3Week 4Example 1PTL (post-run)
M (end of week)PTL (post-run)PTL (post-run)PTL (post-run)Example 2P (idle, no run)PTL (post-run)
WP (idle, no run)M, W (end of week)Example 3P (idle, no run)P (idle, no run)P (idle, no run)
PTL (post-run)M, W (end of week)Example 4P (idle, no run)PTL (post-run)P (idle, no run)PTL (post-run)Example 5P (idle, no run)PTL (post-run)M, W (end of week)P (idle, no run)
PTL (post-run)
Monthly Wash Schedule ScenariosWeek 1Week 2Week 3Week 4Example 1PTL (post-run)
M (end of week)PTL (post-run)PTL (post-run)PTL (post-run)Example 2P (idle, no run)PTL (post-run)
WP (idle, no run)M, W (end of week)Example 3P (idle, no run)P (idle, no run)P (idle, no run)
PTL (post-run)M, W (end of week)Example 4P (idle, no run)PTL (post-run)P (idle, no run)PTL (post-run)Example 5P (idle, no run)PTL (post-run)M, W (end of week)P (idle, no run)
PTL (post-run)
Protocols for the post-run wash, post-run (w/ template line) bleach wash, maintenance wash and standby wash are detailed in the MiSeq System Guide.
?. [Instrument Shutdown & Reboot]
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  and then flip the switch back on. The instrument will boot up the MCS as normal.
?. [Additional Information and Troubleshooting]
Illumina MiSeq Workflow OverviewMiSeq System Denature and Dilute Libraries Guide (15039740)MiSeq System Guide (1000000061014)"Troubleshooting Appendix" - pg. 41 onwardsMiSeq Sample Sheet: Quick Reference Guide (15028392)"Creating a New MiSeq Sample Sheet" (pg. 17-18)MiSeq Performance SpecificationsSequence 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.