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

This article reviews 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).

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

Note: Labs can also consider a shallow shotgun or other well-balanced library spike-in, rather than PhiX, to increase diversity.

• If using this alternate strategy, check index compatibility before adding other libraries.

• Illumina does not support mixing different library types in the same run, so individual evaluation and optimization are required, depending on the library type and size, to make sure each sample receives sufficient reads/coverage.

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 Product Documentation 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 2nM 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 (v/v) 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. Sequence libraries with PhiX spike-in the same day they are diluted.

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].