Prepare the Flow Cell and Libraries

Before loading the flow cell and libraries into the cartridge, bring the flow cell to room temperature, dilute libraries, and optionally spike in PhiX. Libraries are denatured automatically onboard the instrument.

The dilution instructions apply to supported Illumina libraries that are double-stranded. Always perform a quality control analysis, optimize the loading concentration for your library, and use a normalization method that generates double-strand libraries. Bead-based normalization that generates single-stranded libraries is not compatible with onboard denaturation.

Dilute Library to 1 nM

Prepare the flow cell as follows.

Remove a new flow cell from 2°C to 8°C storage.

Set aside the unopened package at room temperature for 10–15 minutes.

Remove Resuspension Buffer (RSB) from -25°C to -15°C storage. Alternatively, use 10 mM Tris-HCl, pH 8.5 in place of RSB.

[Optional] Remove 10 nM PhiX stock from -25°C to -15°C storage.

PhiX is needed only for an optional spike-in or a PhiX-only run.

Thaw RSB and optional PhiX at room temperature for 10 minutes.

In a low-bind microtube, dilute 1 nM library in RSB to the applicable volume:

Library Type	Volume of 1 nM Library (µl)*
100% PhiX (for a PhiX-only run)	12
AmpliSeq Library PLUS for Illumina	7
Illumina DNA Prep	12
Illumina DNA Prep with Enrichment	10
Nextera XT DNA	20
TruSeq DNA Nano	20
TruSeq DNA PCR-Free	12
Illumina DNA PCR-Free Prep	15

* Volumes include overage for accurate pipetting.

Successful sequencing depends on diluting libraries in low-bind microtubes.

Vortex briefly, and then centrifuge at 280 × g for 1 minute.

[Optional] Store 1 nM library at -25°C to -15°C for up to 1 month.

Dilute 1 nM Library to Loading Concentration

In a low-bind microtube, combine the following volumes to prepare 100 µl library diluted to the applicable loading concentration:

Library Type	Loading Concentration (pM)	1 nM Library Volume (µl)	RSB Volume (µl)
100% PhiX (for a PhiX-only run)	100	10	90
AmpliSeq Library PLUS for Illumina	50	5	95
Illumina DNA Prep	100	10	90
Illumina DNA Prep with Enrichment	75	7.5	92.5
Nextera XT DNA	150	15	85
TruSeq DNA Nano	150	15	85
TruSeq DNA PCR-Free	100	10	90
Illumina DNA PCR-Free Prep	120	12	88

These tables provides example loading concentrations. The iSeq 100 is compatible with all Illumina library prep kits except SureCell WTA 3′, but the optimal loading concentration can vary.

Vortex briefly, and then centrifuge at 280 × g for 1 minute.

Set aside diluted library on ice for sequencing. Sequence libraries the same day they are diluted.

If you are not adding PhiX or are performing a PhiX-only run, skip the next section and proceed to Load Consumables Into the Cartridge.

Add a PhiX Control (Optional)

PhiX is a small, ready-to-use Illumina library with balanced nucleotide representation. Adding a 2% PhiX spike-in to your library provides additional metrics. For low-diversity libraries, use a 10% spike-in to increase base diversity.

A spike-in as low as 1% is effective for providing additional metrics, but makes pipetting difficult.

In a low-bind microtube, combine the following volumes to prepare 50 µl 1 nM PhiX:

•

10 nM PhiX (5 µl)

•

RSB (45 µl)

Vortex briefly, and then centrifuge at 280 × g for 1 minute.

[Optional] Store 1 nM PhiX at -25°C to -15°C for up to 1 month.

In a low-bind microtube, combine 1 nM PhiX and RSB to prepare 100 µl PhiX diluted to the same loading concentration as the library.

For example:

PhiX Loading Concentration (pM)	1 nM PhiX Volume (µl)	RSB Volume (µl)
25	2.5	97.5
50	5	95
70	7	93
80	8	92
100	10	90
115	11.5	88.5
200	20	80

Combine PhiX and library:

•

For a 2% spike-in, add 2 µl diluted PhiX to 100 µl diluted library.

•

For a 10% spike-in, add 10 µl diluted PhiX to 100 µl diluted library.

Actual PhiX percentage varies depending on library quality and quantity.

Vortex briefly, and then centrifuge at 280 × g for 1 minute.

Set aside the library with PhiX spike-in on ice.