Onboard Denature and Dilute

This section is only applicable to protocols utilizing XLEAP-SBS chemistry. If your protocol utilizes standard SBS chemistry, refer to Standard SBS Sequencing Protocol.

If using onboard denature and dilute, this step dilutes libraries to the applicable loading concentration. An optional 2% PhiXPhiX is a small, ready-to-use Illumina library with balanced nucleotide representation. spike-in provides additional metrics, base diversity, or a positive control. Increase the PhiX spike-in percentage for libraries with lower base diversity.

If manually denaturing and diluting libraries, refer to Manual Denature and Dilute. This step only applies to onboard denature and dilution.

1. [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.

2. [Optional] Thaw PhiX at room temperature for 5 minutes, and then quantify using a fluorescence-based method, such as Qubit, to confirm PhiX concentration.

If quantification is not possible, proceed with 10 nM concentration.

3. Vortex library or PhiX briefly, and then centrifuge at 280 × g for 1 minute.
4. Using RSB with Tween 20 as diluent, prepare at least 24 µl 2 nM library in a low-bind microtube.

For PhiX spike-in instructions, refer to Add a PhiX Control (Optional).

5. Vortex briefly, and then centrifuge at 280 × g for 1 minute.
1. Combine the following volumes in a low-bind microtube to prepare 24 µl library diluted to the appropriate loading concentration:

Library Type*

XLEAP-SBS Loading Concentration (pM)

XLEAP-SBS 2 nM Library Volume (µl)

XLEAP-SBS RSB With Tween 20 Volume (µl)

Illumina DNA Prep with Enrichment

All: 1000

All: 12

All: 12

Illumina Stranded Total RNA with Ribo-Zero Plus

All: 750

All: 9

All: 15

TruSeq DNA Nano 550

P1/P2 (600-cycle): 1500

P1/P2 (600-cycle): 18

P1/P2 (600-cycle): 6

100% PhiX

P1/P2 (2 x 151 or less cycles): 650

P1/P2 (2 x 300 cycles): 450

P3/P4: 450

P1/P2 (2 x 151 or less cycles): 7.8

P1/P2 (2 x 300 cycles): 5.4

P3/P4: 5.4

P1/P2 (2 x 151 or less cycles): 16.2

P1/P2 (2 x 300 cycles): 18.6

P3/P4: 18.6

* For unlisted library types, start with a loading concentration of 650 pM and optimize over subsequent runs.

This table provides example loading concentrations. The NextSeq 1000/2000 is compatible with all Illumina library preparation kits, but the optimal loading concentration can vary.

2. Vortex briefly, and then centrifuge at 280 × g for 1 minute.
3. Set aside diluted library on ice until ready for sequencing.

Sequence libraries diluted to the loading concentration the same day they are diluted.

4. Proceed as follows.
• If adding PhiX, refer to Add a PhiX Control (Optional).
• If not adding PhiX or are performing a PhiX-only run, refer to Load Consumables Into the Cartridge.
1. Prepare PhiX at the same concentration as the library diluted to the final loading concentration. For example, combine the following volumes in a low-bind microtube to prepare 20 µl PhiX:

Loading concentration (pM)

10 nM PhiX Volume (µl)

RSB with Tween 20 Volume (µl)

750

1.5

18.5

1000

2

18

1500

3

17
2. Combine, for example, the following volumes in a low-bind microtube to prepare 20 µl 1 nM PhiX:
• 10 nM PhiX (2 µl)
• RSB with Tween 20 (18 µl)
3. Vortex briefly, and then centrifuge at 280 × g for 1 minute.
4. Add, for example, 1 µl 1 nM PhiX to 24 µl library diluted to the final loading concentration.

Actual percentage varies depending on library quality and quantity.

5. Set aside the library with PhiX spike-in on ice until ready for sequencing.

Sequence libraries with PhiX spike-in the same day they are diluted.