Instrument washes required for Illumina sequencing platforms

Many different factors contribute to a successful sequencing run. One important aspect is to make sure that the fluidics lines are thoroughly flushed between sequencing runs to prevent any salt accumulation. It is also important that the fluidics systems are well maintained when the instrument is idle. With the introduction of the newer Illumina sequencing platforms, instrument washes are not necessary for iSeq 100 and NextSeq 1000/2000 systems. The new sequencing cartridges contain pumps, valves, and all other fluidics for the system, which are discarded after a run.

This bulletin outlines the types of washes required, the time needed to complete the wash, and how frequently the respective washes should be performed for all Illumina sequencing instruments.

Note: Water and other chemicals used should be lab grade.

MiSeq system

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Manual post-run wash	Standard instrument wash performed between sequencing runs	0.5% Tween 20 wash solution	20 minutes	Perform after every run
Manual post-run wash including template line wash	Post-run wash with user-supplied NaOCl to flush the template line	0.5% Tween 20% and 0.01% NaOCl wash solution	30 minutes	Perform as needed (for VeriSeq PGS runs, wash is performed after every run)
Manual maintenance wash	The wash includes a series of three wash steps that thoroughly flush the system	0.5% Tween 20 wash solution	90 minutes	Perform every 30 days
Manual standby wash	Two consecutive washes that flush each position to remove any remaining reagents or salt accumulation	0.5% Tween 20 wash solution	2 hours	Perform to prepare for idle mode (system unused for ≥ seven days), and every 30 days if the instrument remains idle

Instructions for performing the wash procedures can be found in the MiSeq System Guide.

MiniSeq and NextSeq 500/550 systems

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Manual post-run wash	Standard instrument wash performed between sequencing runs	0.5% Tween 20 wash solution	20 minutes	Perform after every run
Manual post-run wash including template line wash	Post-run wash with user-supplied NaOCl to flush the template line	0.5% Tween 20% and 0.01% NaOCl wash solution	30 minutes	Perform as needed (for VeriSeq PGS runs, wash is performed after every run)
Manual maintenance wash	The wash includes a series of three wash steps that thoroughly flush the system	0.5% Tween 20 wash solution	90 minutes	Perform every 30 days
Manual standby wash	Two consecutive washes that flush each position to remove any remaining reagents or salt accumulation	0.5% Tween 20 wash solution	2 hours	Perform to prepare for idle mode (system unused for ≥ seven days), and every 30 days if the instrument remains idle

Instructions for performing the wash procedures can be found in the MiniSeq System Guide, NextSeq 500 and NextSeq 550 Sequencing System Guide.

HiSeq 1500/2000/2500 Systems (all manual washes)

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Automatic post-run wash	When sequencing is complete, the software initiates an automatic post-run wash using the wash solution and NaOCl provided in the reagent cartridge	Not applicable	60 minutes	Automatic after every run
Manual quick wash	The wash flushes the system with user-supplied Tween 20 wash solution	0.05% Tween 20 wash solution	20 minutes	Required every 14 days (instrument idle with cartridges in place) Required every seven days (instrument is in a dry state with cartridges removed or in Array Mode for NextSeq550) Required after a shutdown
Manual post-run wash	The wash flushes the system with user-supplied NaOCl and Tween 20 wash solution	0.05% Tween 20% and 0.12% NaOCl solution	90 minutes	Required if the automatic post-run wash was not performed

Instructions for performing the wash procedures can be found in the respective system guides here: HiSeq 1500, HiSeq 2000, HiSeq 2500.

HiSeq 3000/4000/X systems (all manual washes)

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Water wash (8 SBS positions)	Required after each sequencing run to wash the system and check fluidics	Laboratory-grade water	20 minutes	Perform after every run If the instrument has been idle for one day or more, perform before beginning a new sequencing run
Water wash (8 SBS and 10 paired-end positions)	Required after each sequencing run to wash the system and check fluidics Recommended for sequencing runs that include an indexing read or are paired end	Laboratory-grade water	60 minutes	Perform after every run If the instrument has been idle for one day or more, perform before beginning a new sequencing run
Water wash (8 SBS, 10 paired-end positions, and 1 template loading position)	Required after each rapid run mode sequencing run to wash the system and check fluidics	Laboratory-grade water	10 minutes	Perform after every run If the instrument has been idle for one day or more, perform before beginning a new sequencing run
Maintenance wash	Two types of workflows: (1) Tween 20 and ProClin 300, or (2) Tween 20 only (if ProClin solution is not available) Either workflow will require a subsequent water wash if the instrument is going to be idle for more than five days	Workflow 1 0.5% Tween 20 wash solution and 0.03% ProClin 300 solution > Workflow 2 0.5% Tween 20 wash solution	130 minutes (for High Output mode), 60 minutes (for Rapid mode)	Perform every 10 days, optionally after a run
Maintenance wash (Switching from High Output to Rapid Run mode)	Requires performing a Rapid Mode maintenance wash (assuming that the maintenance wash in High Output mode has already been performed) Two types of workflows: (1) Tween 20 and ProClin 300, or (2) Tween 20 only (if ProClin solution is not available) Either workflow will require a subsequent water wash if the instrument is going to be idle for more than five days	Workflow 1 0.5% Tween 20 wash solution and 0.03% ProClin 300 solution Workflow 2 0.5% Tween 20 wash solution	60 minutes	Perform when switching modes from High Output to Rapid Run
Maintenance wash (Switching from Rapid Run to High Output mode)	Requires performing a Rapid Mode maintenance wash followed by a maintenance wash in High Output mode Two types of workflows: (1) Tween 20 and ProClin 300, or (2) Tween 20 only (if ProClin solution is not available) Either workflow will require a subsequent water wash if the instrument is going to be idle for more than five days	Workflow 1 0.5% Tween 20 wash solution and 0.03% ProClin 300 solution Workflow 2 0.5% Tween 20 wash solution	60 minutes + 130 minutes = 190 minutes	Perform when switching modes from Rapid Run to High Output

Instructions for performing the wash procedures can be found in the respective system guides here: HiSeq 3000, HiSeq 4000, HiSeq X.

cBot 2 System (all manual washes)

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Water wash (8 SBS positions)	Required after each sequencing run to wash the system and check fluidics	Laboratory-grade water	20 minutes	Perform after every run If the instrument has been idle for one day or more, perform before beginning a new sequencing run
Water wash (8 SBS and 10 paired-end positions)	Required after each sequencing run to wash the system and check fluidics Recommended for sequencing run that includes an indexing read or paired end	Laboratory-grade water	60 minutes	Perform after every run If the instrument has been idle for one day or more, perform before beginning a new sequencing run
Maintenance wash	Two types of workflows: (1) Tween 20 and ProClin 300, or (2) Tween 20 only (if ProClin solution is not available) Either workflow will require a subsequent water wash if the instrument is going to be idle for more than five days	Workflow 1 0.5% Tween 20 wash solution and 0.03% ProClin 300 solution Workflow 2 0.5% Tween 20 wash solution	90 minutes	Perform every 10 days, optionally after a run

Instructions for performing the wash procedures can be found in the cBot 2 System Guide.

NovaSeq 6000 systems

Type of wash	Description	Reagents to prepare	Approximate time	Frequency
Pre-run and post-run water wash	Perform an instrument wash before and after each run to clear salts and enzymes from the instrument hardware and to prevent clogs	Laboratory-grade water		Between each run and if the instrument is idle for more than one day
Maintenance wash	Perform wash to remove traces of reagents from internal cBot components and inhibit the growth of microorganisms	5% DECON or 100 mM NaOH (if DECON is not available)		Perform once per month

Instructions for performing the wash procedure can be found in the NovaSeq 6000 System Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #5603), contact Illumina Technical Support [e-mail].