Recommendations for sequencing Illumina 16S libraries with the NextSeq 1000/2000 600 cycle kits

This article discusses how to sequence 16S libraries prepared with the Illumina 16S demonstrated protocol using the 600 cycle kits, now available for the NextSeq 1000/2000.

For 16S libraries prepared with the Illumina 16S demonstrated protocol, Illumina development has tested a loading concentration of 1000 pM with a 40% PhiX spike-in (volume/volume, v/v) with Standard SBS reagents.

Each lab may need to further adjust the loading concentration and PhiX spike-in to maximize data output and quality.

NextSeq 1000/2000 sample loading

Final 16S libraries are typically concentrated (have high yields) so the Onboard Denature and Dilute protocol for the NextSeq 1000/2000 can be used. See the NextSeq 1000/2000 Denature and Dilution protocol generator for the detailed protocol.

Below is an overview of the dilution steps prior to loading and onboard denaturation.

Note: This article does not discuss manual denature and dilution.

Before starting

Quantify the PhiX control.

• Remove 10 nM PhiX stock from -25°C to -15°C storage.

• Thaw PhiX at room temperature for 5 minutes or until completely thawed, vortex well, and then quantify using a fluorescence-based method to confirm PhiX concentration.

1. Dilute 16S Library to 2 nM. Using RSB with Tween 20 as diluent, prepare at least 24 µl 2 nM library in a low-bind microtube.

2. Dilute 2 nM 16S Library to Loading Concentration. To reach a loading concentration of 1000 pM, combine 12 µl of 16S libraries at 2 nM concentration with 12 µl of RSB with Tween 20.

3. Prepare PhiX at the same concentration as the library diluted to the final loading concentration. Note: If the 16S library is loaded at 1000 pM, the PhiX also needs to be at 1000 pM. If using a different 16S library pool concentration, adjust the PhiX concentration accordingly.

• Combine 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)

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

• Adjust volumes as needed if using a different concentration.

1. Combine 16S libraries and PhiX.

The example below shows calculation for 40% PhiX spike in to create 25 µl pool (20 µl of pool will be loaded). Adjust if targeting a different spike in percentage.

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

Demo data For demo sequencing run performance and data analysis, see the following (BaseSpace Sequence Hub login required for access).

NextSeq2000: 16S amplicons on P1 600 cycles kit Demo Run Demo Project

NextSeq2000: 16S amplicons with Illumina DNA Prep on P1 600 cycles kit Demo Run Demo Project

For any feedback or questions regarding this article (Illumina Knowledge Article #7461), contact Illumina Technical Support [e-mail].