TITLE: Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing
AUTHORS: Julie Haendiges, Ruth Timme, Maria Balkey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure outlines the procedures for operation and maintenance of an Illumina MiSeq Sequencer for Whole Genoem Sequencing.</div><div class = "text-block">This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.</div><div class = "text-block"><span style = "font-weight:bold;">Complete in order:</span></div><div class = "text-block"><span style = "font-weight:bold;">1. DNA Extraction (Manual DNA Extraction </span><span style = "font-weight:bold;font-style:italic;">or</span><span style = "font-weight:bold;"> Automated DNA Extraction using the Qiacube)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">2. DNA Quantitation</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">2. DNA Quantitation</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library Preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><span style = "font-weight:bold;">4. Sequencing using Illumina MiSeq (Included SOP)</span></div><div class = "text-block"><span style = "font-weight:bold;">5. Data Quality Checks and NCBI Submission</span></div></div>

[STEPS]
?. [Starting the Illumina MiSeq Desktop Sequencer]
Power on the instrument using the power switch located on the back of the instrument.
?. [Starting the Illumina MiSeq Desktop Sequencer]
If the MiSeq is networked, this is the point where the username and password would be entered.
?. [Starting the Illumina MiSeq Desktop Sequencer]
After the MiSeq computer has booted up, the MiSeq Control Software (MCS) will load automatically. Wait for the machine to complete initialization.
?. [Preparing the Reagent Cartridge for the MiSeq]
Plan for sufficient time to allow complete thawing of the cartridge. The Cartridge and HT1 are contained in the same box and must be thawed on ice or at 2-8oC.See the table below for guidance regarding reagent kit thawing and storage:
?. [Preparing the Reagent Cartridge for the MiSeq]
Once thawed, invert the reagent cartridge 10 times to mix, and then visually inspect to verify that all reagents are completely thawed and free of participates.
?. [Preparing the Reagent Cartridge for the MiSeq]
Gently tap the cartridge on the bench to dislodge air bubbles in the bottom of the wells, and wipe the cartridge off with a paper towel.
?. [Preparing the Reagent Cartridge for the MiSeq]
Place the thawed reagent cartridge and HT1 on ice or at 2-8oC until the sample is ready to be loaded. (The cartridge contains photosensitive reagents and should be kept in the dark)
?. [Setting up the run on the MiSeq]
Prior to setting up the run, ensure that there is enough free disk space (100GB) on the instrument. If there is less than 100GB available, proceed to Instrument Mainentance section to free up disk space.
?. [Setting up the run on the MiSeq]
From the MCS home screen, select Sequence
?. [Setting up the run on the MiSeq]
Select a run option:    • Local Run Manager: Select this option if the run has previously been set up in the LRM Software    • Sample Sheet: Select this option if a sample sheet has been generated for the run. The sample sheet will be sent         to the LRM for validation andautomatic run creation    • Manual: Create a run manually but selecting all parameters of the run. No secondary analysis will be performed if this option is selected.
?. [Setting up the run on the MiSeq]
Enter BaseSpace email and password if applicable (Note: DM does not use BaseSpace). Select Next.
?. [Setting up the run on the MiSeq]
Select the Local Run Manager run or select the sample sheet for the run depending on the option used in Step 10.
?. [Setting up the run on the MiSeq]
Proceed immediately to Preparing and Loading the Flow Cell section.
?. [Preparing and Loading the Flow Cell]
The flow cell and Incorporation buffer (PR2) are located in Box 2 of 2 and stored at 2-8oC.
?. [Preparing and Loading the Flow Cell]
While wearing clean, powder-free gloves, carefully remove the flow cell from the container, by gripping the base of the plastic end.
?. [Preparing and Loading the Flow Cell]
Lightly rinse the flow cell with molecular-grade water, making sure to rinse both sides of the glass and plastic casing. It is very critical to ensure all excess salts are removed. If salts dry in the imaging area, imaging can be affected during the run.
?. [Preparing and Loading the Flow Cell]
Pat dry the flow cell using a lint-free wipe, using care around the black port gasket.
?. [Preparing and Loading the Flow Cell]
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 fibers. An alcohol wipe can also be used for this step. Do not add ethanol directly to the flow cell, and avoid getting ethanol on the port gasket.
?. [Preparing and Loading the Flow Cell]
Dry any excess ethanol with the lens paper and visually inspect to make sure that the flow cell is free of smudges or debris and dry. Inspect the port gasket to ensure it 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 gently press the release button to open the clamp and remove the previously used flow cell.
?. [Preparing and Loading the Flow Cell]
Holding the flow cell by the edges, place it on the flow cell stage.
?. [Preparing and Loading the Flow Cell]
Gently press down on the flow cell clamp to close it over the flow cell (it will click when closed fully). Close the compartment door. The software should identify the flow cell RFID.
?. [Preparing and Loading the Flow Cell]
Select Next.
?. [Loading the Incorporation Buffer]
Gently invert the bottle of PR2 to mix, and then remove the lid.
?. [Loading the Incorporation Buffer]
Open the reagent compartment door.
?. [Loading the Incorporation Buffer]
Raise the sipper handle until it locks into place.
?. [Loading the Incorporation Buffer]
Remove the wash bottle and load the PR2 bottle. Re-cap the wash bottle and set aside for future use.
?. [Loading the Incorporation Buffer]
Empty the contents of the waste bottle into the appropriate chemical waste container.
?. [Loading the Incorporation Buffer]
Lower the sipper handle, make sure the sipper lowers into the PR2 and waste bottles.
?. [Loading the Incorporation Buffer]
The software will identify the RFID of the PR2 bottle.Note: If the RFID is not read by the system for any step, the software prompts you through the steps to obtain a temporary bypass code and proceed with setting up the run. For more information, see “Resolving RFID Read Failure” in the MiSeq System User Guide.
?. [Loading the Incorporation Buffer]
Select Next.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Remove the thawed reagent cartridge, ensuring that it has been thoroughly dried and the reagents have been mixed by inversion.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Tap the reagent cartridge on a hard surface to collect contents at the bottom of the reservoirs.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Use a clean 1000 μl pipette tip to pierce the foil seal over the reservoir labeled Load Sample.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Transfer  of the denatured DNA library into the Load Sample reservoir, take care to avoid droplets on the foil seal as sample is being transferred.
600 µl
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Check the bottom of the cartridge to ensure there are no bubbles present at the bottom of the reservoirs. If so, tap the cartridge gently on the tabletop to release the bubbles.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Make sure the MCS screen states Load Reagent Cartridge to ensure the sippers in the chiller have been raised.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Open the reagent chiller door, remove the wash tray, and dry bottom of chiller compartment if necessary. (Humidity fluctuations in the laboratory can cause condensation to pool in the chiller)
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Hold the reagent cartridge by the end with the Illumina label and slide it into the chiller until it stops.Note: Do not leave the chiller door open for extended periods of time
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Close the chiller door and check the screen to confirm the software has identified the RFID of the reagent cartridge.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Close the reagent compartment door.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Select Next. Review run parameters and ensure that they are correct. Select Next to start the Pre-Run Check.Note: The system performs a check of all run components, disk space, and network connections prior to a run start. If any part of the pre-run check fails, a message will appear with general instructions describing the error and detailshow to correct it. For more information, see “Resolving Run Setup Errors” in the MiSeq System User Guide. If all items successfully pass the pre-run check, the system is ready to start the run.
?. [Loading the Sample into the Cartridge and Loading into the Instrument]
Select Start Run. Once the run has started, do not open the instrument or touch the instrument monitor unless to pause or stop the run.Note: The MiSeq is sensitive to vibration, performing tasks that cause vibration during a run could cause the run to fail or adversely impact sequencing results.
?. [Post-Run Washes and Reagent Disposal]
Post-Run washes are required at the conclusion of every run. It is recommended by FDA-GenomeTrakr to also perform a post-run wash (Tween only) at the beginning of each run.
?. [Post-Run Washes and Reagent Disposal]
Once the run is complete, select Start Wash option at the bottom of the MCS.
?. [Post-Run Washes and Reagent Disposal]
Check the box for Perform optional template line wash to proceed with a Postrun bleach wash. The purpose of the bleach wash is to reduce the rate of carryover of nucleic acids to proceeding runs.
?. [Post-Run Washes and Reagent Disposal]
Prepare fresh wash solution with Tween-20 and laboratory grade water:
?. [Post-Run Washes and Reagent Disposal]
Prepare a fresh dilution of molecular-grade sodium hypochlorite (NaOCl – Bleach), using molecular-grade water.
?. [Post-Run Washes and Reagent Disposal]
Insert the MiSeq wash tube with the 0.01% NaOCl solution into position 17 of the wash tray until the neck of the tube is flush with the tray. The tube must be put into position 17 only. Inserting the tube in other positions can cause cluster generation failure and can damage the fluidic system.
?. [Post-Run Washes and Reagent Disposal]
Add at least  of the 0.5% Tween-20 Wash solution in to all other reservoirs of the wash tray.
6 µl
?. [Post-Run Washes and Reagent Disposal]
Add  of 0.5% Tween-20 Wash solution to the 500 ml wash bottle.
350 µl
?. [Post-Run Washes and Reagent Disposal]
Open the reagent compartment door and reagent chiller door, and remove the used reagent cartridge from the chiller.
?. [Post-Run Washes and Reagent Disposal]
Slide the wash tray into the chiller until it stops, then close the chiller door.
?. [Post-Run Washes and Reagent Disposal]
Raise the sipper handle fully and remove the used PR2 bottle and replace with the wash bottle. Discard the residual buffer in chemical waste and the used PR2 bottle.
?. [Post-Run Washes and Reagent Disposal]
Remove waste bottle and discard of the contents in a chemical waste receptacle that is appropriate for formamide. Return the waste bottle to the reagent compartment.
?. [Post-Run Washes and Reagent Disposal]
Lower the sipper handle and close the reagent compartment door.
?. [Post-Run Washes and Reagent Disposal]
Select Next
?. [Post-Run Washes and Reagent Disposal]
Once the post-run bleach wash is complete, select Done on the screen to return to the MiSeq Control Software home screen. Leave the used flow cell, wash tray, and wash bottle on the instrument. The sippers will remain in the down position, they should remain in the wash solution to prevent drying out and air from entering the fluidics. OPTIONAL: A second-post run wash can be performed with Tween-20 wash solution only to clear out any excess NaOCl from the lines.
?. [Post-Run Washes and Reagent Disposal]
10 % Tween-20: Add  of Tween-20 to of molecular grade water.
5 µl
45 µl
?. [Post-Run Washes and Reagent Disposal]
0.5% Tween-20 Wash: Add  of 10% Tween-20 to  of moleculargrade water.
25 µl
475 µl
?. [Post-Run Washes and Reagent Disposal]
Invert five times to mix
?. [Post-Run Washes and Reagent Disposal]
Preparing stock solutions of 10% and 0.5% tween are not advised, these stocks can grow mold and become contaminated.
?. [Post-Run Washes and Reagent Disposal]
0.2% NaOCl: Add  of 5% NaOCl to  of molecular-grade water.
36 µl
864 µl
?. [Post-Run Washes and Reagent Disposal]
0.01% NaOCl: Add  of the 0.2% NaOCl to  of molecular-grade water.
50 µl
950 µl
?. [Post-Run Washes and Reagent Disposal]
Add the entire 1.0mL of the 0.01% NaOCl to the wash tube.Note: Using the correct concentration of NaOCl is important. If the concentration is too high, it can cause cluster generation failures in subsequent runs. It is recommended to purchase the NaOCl from Sigma as listed in the reagents section. Use of Clorox is not advised due to the variability in concentration of NaOCl. Template line washes should not be performed with maintenance or standby washes.
?. [Instrument Maintenance]
Maintenance during Normal Operation:
?. [Instrument Maintenance]
Maintenance during Idle Operation: (Instrument unused for >14 days):
?. [Instrument Maintenance]
Perform a Post-Run Wash (without bleach):
?. [Instrument Maintenance]
It is a good practice to run a Post-Run wash prior to starting a run to ensure that the fluidic lines are flushed and there are no air bubbles that could affect the run. A post-run wash should also be performed once a week if the instrument is sitting idle.
?. [Instrument Maintenance]
Make sure a flow cell is loaded on the instrument. From the MCS home screen, select Perform Wash. From the Perform Wash screen, select Perform Post-Run Wash.
?. [Instrument Maintenance]
Prepare fresh 0.5% Tween-20 wash solution and add  to each reservoir in the wash tray, and  to the wash bottle.
6 µl
350 µl
?. [Instrument Maintenance]
Load the wash tray and wash bottle onto the instrument.
?. [Instrument Maintenance]
Select Next, the wash will begin.
?. [Instrument Maintenance]
When the wash is complete, leave the used flow cell, wash tray, and wash bottle containing the remaining wash solution on the instrument.
?. [Instrument Maintenance]
Perform a Maintenance Wash:
?. [Instrument Maintenance]
Maintenance washes are performed every 30 days to ensure optimal performance. The wash takes approximately 90 minutes to complete. The wash includes a series of three washes that thoroughly flush the system.Note: The MCS may lock down the instrument if a maintenance wash is not performed and will not allow a run to start until it is performed.
?. [Instrument Maintenance]
Make sure a flow cell is loaded on the instrument. From the MCS home screen, select Perform Wash. From the Perform Wash screen, select Perform Maintenance Wash.
?. [Instrument Maintenance]
Prepare fresh 0.5% Tween-20 wash solution and add  to each reservoir in the wash tray, and  to the wash bottle.
6 µl
350 µl
?. [Instrument Maintenance]
Load the wash tray and wash bottle onto the instrument.
?. [Instrument Maintenance]
Select Next, the first wash will begin.
?. [Instrument Maintenance]
Always use fresh wash solution for each wash step. Reusing wash solution from previous washes can return waste into the fluidics.
?. [Instrument Maintenance]
Fill the second wash tray with fresh 0.5% Tween-20 wash solution.
?. [Instrument Maintenance]
When the instrument prompts to, remove the first wash tray and wash bottle and replace with the second wash tray and wash bottle.
?. [Instrument Maintenance]
Select Next, the second wash will begin.
?. [Instrument Maintenance]
Dispose of the waste from the first wash tray and wash bottle.
?. [Instrument Maintenance]
Fill the first tray with fresh 0.5% Tween-20 wash solution.
?. [Instrument Maintenance]
When the instrument prompts to, remove the second wash tray and wash bottle and replace with the first wash tray and wash bottle.
?. [Instrument Maintenance]
Select Next, the final wash will begin.
?. [Instrument Maintenance]
When the wash is complete, leave the used flow cell, wash tray, and wash bottle containing the remaining wash solution on the instrument.
?. [Instrument Maintenance]
Instrument Reboot:
?. [Instrument Maintenance]
The instrument should be rebooted weekly and after file deletion to free up virtual memory.
?. [Instrument Maintenance]
On the MCS home screen, select Manage Instrument then Reboot. It will take approximately 10 minutes for the system to reboot and the MCS to restart.
?. [Instrument Maintenance]
Alternatively, the instrument can be power cycled by selecting the Manage Instrument menu option and then Shut Down. Once the instrument has shut down, reach behind the right-hand side of the instrument and flip the power switch located near the power cord. Allow for at least 1 minute before turning the power back on.
?. [Instrument Maintenance]
Management of Disk Space on the Data (D:\) Drive:
?. [Instrument Maintenance]
The MiSeq requires 100GB of free disk space on the D:\ drive before starting a sequencing run. Data should be cleared only after any data transfer, and if no troubleshooting is necessary for that run. The following outlines whichfiles can be deleted and their locations:
?. [Instrument Maintenance]
Navigate to Data (D:/Illumina/MiSeqOutput/(most recent run folder)
?. [Instrument Maintenance]
Navigate to D:/Illumina/MiSeqAnalysis/Note: It is suggested to reboot the instrument following data deletion in order to free virtual memory in the system.
?. [Instrument Maintenance]
Perform a Standby Wash:
?. [Instrument Maintenance]
If the instrument is not to be used for an extended period of time (>14 days), the instrument should be prepared for idle mode by performing a standby wash. The standby wash takes approximately 2 hours to complete.
?. [Instrument Maintenance]
The standby wash consists of two 60 minutes washes with the 0.5% Tween-20 wash solution.
?. [Instrument Maintenance]
After the initial standby wash, a standby wash needs to be performed every 30 days while the instrument remains idle.
?. [Instrument Maintenance]
To bring an instrument out of idle mode, a maintenance wash must be performed before a run can be initiated.
?. [Forms]
MiSeq Maintenance Records:
?. [Appendix 1: Addition of PhiX Control]
PhiX is a balanced genome of a bacteriophage and recommended by Illumina as an internal control to assess how well the sequencing instrument is performing. A PhiX run is recommended after any instrument repair or service. PhiX can also be spiked into a final library as a control or to improve sequencing of low-diversity libraries.
?. [Appendix 1: Addition of PhiX Control]
Diluted Denatured PhiX Library Preparation:
?. [Appendix 1: Addition of PhiX Control]
The denatured PhiX control should be diluted to 20 pM, when used with v3 chemistry and 12.5 pM, when used with v2 chemistry for optimal cluster density.
?. [Appendix 1: Addition of PhiX Control]
Prepare 0.2 N NaOH and place on ice until ready to use.
?. [Appendix 1: Addition of PhiX Control]
Combine  of 10 nM PhiX library from the stock and  of EBT to obtain 4 nM PhiX library in a new 1.5 ml microcentrifuge tube.
2 µl
3 µl
?. [Appendix 1: Addition of PhiX Control]
Combine  of 4 nM PhiX Library and of 0.2 N NaOH and mix well.
5 µl
5 µl
?. [Appendix 1: Addition of PhiX Control]
Incubate for at room temperature to denature the PhiX library.
5 µl
?. [Appendix 1: Addition of PhiX Control]
IMMEDIATELY add  of pre-chilled HT1 to the tube containing the 10 μl ofdenatured PhiX to obtain 20 pM of denatured Phix library.
990 µl
?. [Appendix 1: Addition of PhiX Control]
Label, date, and initial the tube. Denatured 20 pM PhiX libraries may be stored up to 3 weeks at -15oC to -25oC.
?. [Appendix 1: Addition of PhiX Control]
If you are using a MiSeq Reagent v3 Kit, no further dilution is required. If you are using a v2 kit, then remove 375 μl from the 20 pM PhiX dilution and add 375 μl of HT1 back into this tube. The result is 1 ml of 12.5 pM PhiX library.
?. [Appendix 1: Addition of PhiX Control]
PhiX Spike-in Control:
?. [Appendix 1: Addition of PhiX Control]
A stock of diluted PhiX can be prepared and stored for subsequent use.
?. [Appendix 1: Addition of PhiX Control]
Prepare a 20 pM of denatured PhiX as described in steps 61.1 - 61.7.
?. [Appendix 1: Addition of PhiX Control]
Dilute the denatured 20 pM PhiX library to 8 pM by adding  of library to of pre-chilled HT1.
40 µl
60 µl
?. [Appendix 1: Addition of PhiX Control]
Determine the percentage of PhiX (N%) to add to run. If troubleshooting a library preparation, then 1 – 10% is appropriate. If using PhiX routinely in every run, then 1% is recommended, for low-diversity libraries use of a 5% spike-in is recommended.
?. [Appendix 1: Addition of PhiX Control]
For a 1% PhiX spike, add 10 μl of 8 pM PhiX to 990 μl of the final denatured 10-20 pM sample library. For other percentages, combine N x 10 μl of 8 pM PhiX with (1000 – (N x 10) μl of sample library where N = the percentage of spike-in.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Exporting a MiSeq Sample Sheet from the GenomeTrakr Flex Library Workbook:
?. [Appendix 2: MiSeq Sample Sheet Setup]
Ensure that the SampleSheet tab is properly populated. (Do not modify the Indices tab, the index sequences are populated from this tab)
?. [Appendix 2: MiSeq Sample Sheet Setup]
Save as a .csv or export the tab as a .csv:To Save As:Open the File menu option and select Save AsChange Save as type: to CSV (Comma delimited)Navigate to the desired file folder location and save the file using the laboratory Plate ID as the filenameClick OK to save only the active sheet, and Yes in the following window to keep using the CSV format.ORTo export as .csv:Open the File menu option and select ExportSelect Change File TypeChoose CSV (Comma delimited)Save and select OK to save only the active sheetORTo export as .csv:Open the File menu option and select ExportSelect Change File TypeChoose CSV (Comma delimited)Save and select OK to save only the active sheet
?. [Appendix 2: MiSeq Sample Sheet Setup]
Transfer this file to the Sample Sheet folder on the MiSeq, or a shared network drive that can be accessed by the MiSeq.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Open the Local Run Manager software.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select +Create Run.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select Import Sample Sheet.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Verify the information that is populated is correct.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select Save Run.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Manually Entering Run Information into Local Run Manager:
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select +Create Run.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select Generate Fastq.
?. [Appendix 2: MiSeq Sample Sheet Setup]
Enter information for the following required fields:Run NameLibrary Prep Kit – (Nextera XT or Nextera DNA CD Indexes – 96 Indexes Plated)Read Type – Paired EndRead Length – 151 or 251 (Fill in both R1 and R2)Adapter Trimming – ON
?. [Appendix 2: MiSeq Sample Sheet Setup]
Enter the # of rows to be added to the sample sheet.NOTE: The two required fields are Sample ID and Index Well
?. [Appendix 2: MiSeq Sample Sheet Setup]
Select Save Run.
?. [Reference Documents]
MiSeq System Guide (August 2019)MiSeq Denature and Dilute Libraries Guide (February 2019)Local Run Manager Software Guide v1 (January 2020)Local Run Manager Software Guide v2 (January 2020)