When returning to the lab after an extended shutdown or inactivity,
it is important to make sure all lab resources are up and running and
ready to be used. Below are best practices to check library prep,
sequencing and microarray reagents and instruments to ensure a
Laboratory and Reagent Checks | Sequencing Instrumentation Considerations |
Library Preparation Considerations | Infinium™ Assay Considerations
Laboratory and Reagent Checks
Wipe down all lab surfaces with 10% bleach to remove any
accumulated dust or contaminants.
- Pay particular attention to typical ‘hot spots’:
- Benchtops used for processing amplified DNA
- Refrigerator and freezer door handles
- Computer keyboard and mouse
vortexers, and thermal cyclers
- Clean all
equipment in any pre-amp areas (including pipettes)
to the bulletin Impact
of ammonium-based cleaning products on sequencing run
performance for additional details
Proper storage and handling of reagents, kits, and user-supplied
materials should be followed according to documented procedures.
Check connectivity to network drives or servers used for file
storage (output run folders, manifest files, cluster files, DMAPs) or
data processing (IDAT repository).
Sequencing Instrumentation Considerations
Instrument System Guides: iSeq™ 100
| NextSeq™ 500/550
| NextSeq™ 1000/2000
| HiSeq™ 3000/4000
| HiSeq™ X
| NovaSeq™ 6000
For instruments that were left on:
- Confirm the recommended maintenance/manual/standby washes
were performed per the appropriate system guide.
- Perform a
full power cycle, following the steps in the system guide.
- Leave system off for at least 5 minutes before powering back
Note: While the iSeq™ 100 and NextSeq™ 1000/2000 do
not have onboard fluidics, a power cycle is recommended for these
instruments as well.
For instruments that were turned off:
- Power on the instrument and make sure that the control
- Perform two maintenance/manual washes
to rehydrate the fluidics system (excluding iSeq 100 and NextSeq
Note: If an additional maintenance/manual/standby wash using
laboratory-grade water was not performed prior to instrument shutdown,
perform three maintenance/manual washes to rehydrate the fluidics
system, then run System Checks to test the fluidics delivery. System
Check details are found in the appropriate system guides.
Library Preparation Considerations
Starting a New Library Prep
When starting new library preparation, Illumina recommends
reviewing the best practices for the specific library prep kit being
used. This information is found on the appropriate product support
Proceeding to a Sequencing Run with Stored Libraries
For any Illumina library that has been stored for longer than the
recommended time, as specified in the reference guide, Illumina
recommends repeating quality control (QC) and quantification prior to
sequencing. Re-QC is beneficial because libraries can degrade during
prolonged storage, leading to loss of overall yield. Recommended QC
methods for most Illumina libraries are summarized in the Library
quantification and quality control quick reference guide support
For libraries requiring bead-based
normalization, Illumina recommends storing the library at the last
safe stopping point, just prior to the bead-based normalization step,
as specified in the protocol guide. Libraries stored as double
stranded DNA (dsDNA, before bead-based normalization) are more stable
than libraries stored as single stranded DNA (ssDNA, after bead-based
normalization). If a library has been stored long term after
bead-based normalization, Illumina recommends repeating the bead-based
normalization of the original dsDNA library.
Infinium™ Assay Considerations
Te-Flow and Water Circulator:
- Confirm that there is no microbial growth in the
water circulator and that the water is at the proper fill level.
- If microbial
growth is present, follow the instructions in the Water
Circulator Manual (pg. 57 — 60) for cleaning the water
circulator and refresh with algicide if needed.
- Do not
use chlorine bleach to clean the water circulator.
- Check the waste tubing lines of the Te-Flow and white
reagent pan. Bleach can be squirted through the waste tubing lines
leading to the waste carboy to clean them, if needed.
the waste carboy has been emptied or moved, make sure it is back in
its proper position, with an unimpeded, gravity-driven flow of waste
from the Te-Flow Reagent pan to the waste carboy. Make sure that
waste tubing is not kinked and there are no ‘uphill’ runs to the
Tecan/Illumina Automated Pipetting System (IAPS):
- If the Tecan fluidics were stored dry, refill the system
- Empty the waste liquid container and
disposable tip waste bag, if necessary.
- Make sure that the
Te-Flow is seated correctly in the pan, and that the Te-Flow is in
the appropriate location on the Tecan deck.
- Power on the
- Refer to the following sections in the Infinium
Assay Lab Setup and Procedures Guide under Robot Usage and
Preparing the Infinium Automated Pipetting System for Use
(perform at least two bleach washes)
Note: if the system was idle for a long time and/or
dried out prior to shut down, multiple System Wash cycles
are needed to purge all air from the fluidics.
Testing Volume Accuracy of the IAPS Tips
- Power on the scanner and make sure that the Archimedes drive
appears on the PC before launching ICS software.
- Clean the
BeadChip carriers and iScan Nest (tray that carrier is placed on)
with an Ethanol wipe to remove any dust or built up residue.
- Turn on the scanner at least 30 minutes before use to give the
lasers time to warm up.