> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000007461.md).

# 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](https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html) 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](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=nextseq1k2k) 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.

4. 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.

![](/files/SruOSuBmyUiIqCZM6Tdd)

5. 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](https://ilmn-sso.basespace.illumina.com/s/wKSMrQrfMvIZ)\
[Demo Project](https://ilmn-sso.basespace.illumina.com/s/bcCnMT4bIj6P)

**NextSeq2000: 16S amplicons with Illumina DNA Prep on P1 600 cycles kit**\
[Demo Run](https://ilmn-sso.basespace.illumina.com/s/nT22kKqm0Icj)\
[Demo Project](https://ilmn-sso.basespace.illumina.com/s/VXIBSVVBLpwL)

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #7461), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000007461%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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