16S library preparation for the MiSeq i100 Series 25M Reagent Kit (1000 cycles)

Illumina has released the MiSeq i100 Series 25M Reagent Kit (1000 cycles) to enable 2x501 bp sequencing on the MiSeq i100.

The following provides information for this application of Illumina technology that has been demonstrated internally and may be of interest to users. This information is provided as‐is, is not an official Illumina protocol, and is not accompanied by any rights or warranties. Users of this information should obtain any licenses required and materials from authorized vendors. Illumina products mentioned herein are for research use only. While feedback is welcomed, this application is not supported by Illumina Technical Support and Field Application Scientists.

Illumina has tested three 16S target amplicons, one spanning the V1-V5 regions, one spanning the V1-V6 regions, and one spanning the V1-V9 regions for sequencing on the MiSeq i100 Series instruments with a 2x501 bp read configuration. Libraries are prepared with a two-step PCR process. The first PCR step targets the locus for amplification and adds partial adapter sequences. The second PCR step uses the Illumina DNA/RNA UD index primers to uniquely index the samples and complete the adapter sequences required for sequencing.

Targeting primers Illumina has separately tested the following three primer pairs. The gene‐specific sequences used in this protocol target variable regions within the 16S gene; the amplicon target can be selected based on experimental and data needs. The locus specific primers used in this protocol are from Klindworth, et al.* Partial Illumina adapter overhang sequences are added to the gene‐specific sequences. The full-length primer sequences, using standard IUPAC nucleotide nomenclature, are below. * Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies Klindworth, et al. (2013) PMCID: PMC3592464 PMID: 22933715 https://pmc.ncbi.nlm.nih.gov/articles/PMC3592464/

Illumina recommends using standard desalting purification when ordering oligo primer sets.

Note: the following protocol uses one primer pair, not a mixture of these pairs.

For targeting the 16S V1-V5 region, Illumina has tested the following primer pair. This pair generates an amplicon of ~919 bp, allowing for overlap between the paired end sequencing reads with the 2x501 bp read configuration. The locus-specific region is bolded.

• 16S Amplicon PCR Forward Primer 27F 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTYGATYMTGGCTCAG

• 16S Amplicon PCR Forward Primer 907R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGTCAATTCMTTTGAGTTT

For targeting the 16S V1-V6 region, Illumina has tested the following primer pair. This pair generates an amplicon of ~1057 bp; there is no overlap between the paired end sequencing reads with the 2x501 bp read configuration. The locus-specific region is bolded.

• 16S Amplicon PCR Forward Primer 27F 5’ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTYGATYMTGGCTCAG

• 16S Amplicon PCR Forward Primer 1027R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGACRRCCATGCANCACCT

For targeting the 16S V1-V9 region, Illumina has tested the following primer pair. This pair generates an amplicon of ~1501 bp; there is no overlap between the paired end sequencing reads with the 2x501 bp read configuration. The locus-specific region is bolded. Note: Illumina has tested this primer pair and has successfully generated sequencing libraries, though sequencing performance tested is limited and Illumina is unable to provide support or guidance for sequencing this amplicon target at this time.

• 16S Amplicon PCR Forward Primer 27F 5’ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTYGATYMTGGCTCAG

• 16S Amplicon PCR Forward Primer 1492R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNTACCTTGTTACGACT

Input Illumina has tested using 50 ng input of a control bacterial community. Input may need to be optimized based on sample complexity, presence of host/non-target DNA, and experimental needs.

Amplicon PCR This step uses PCR to amplify the target amplicon out of a DNA sample using region of interest‐specific primers with overhang adapters attached.

Reagents:

• Input/template DNA

• Forward primer (10 µM)

• Reverse primer (10 µM)

• 2X NEB Phusion High Fidelity PCR Master Mix with HF Buffer (NEB #M0531)

• DMSO

• Nuclease-free water

Protocol:

Set up the following reaction in a PCR plate.

Reagent	Volume
Input DNA	10 µl
Forward primer (10 µM)	2.5 µl
Reverse primer (10 µM)	2.5 µl
2X NEB Phusion MasterMix	25 µl
DMSO	1.5 µl
Nuclease-free water	8.5 µl
Total	50 µl

Pipette reactions to mix well.

Seal plate and perform PCR in a thermal cycler using the following program:

Preheat lid to 100°C. 98°C for 30 seconds 25 cycles of: — 98°C for 10 seconds — 55°C for 15 seconds — 72°C for 20 seconds 72°C for 5 minutes Hold at 4°C

Amplicon PCR QC Users may perform QC of the Amplicon PCR product using a Bioanalyzer, TapeStation, Fragment Analyzer, or equivalent to assess amplification and the presence of possible off-target products.

Example TapeStation trace of the V1-V5 amplicon

Example TapeStation trace of the V1-V6 amplicon

Example TapeStation trace of the V1-V9 amplicon

Amplicon PCR Clean‐Up Illumina performed testing for this protocol without performing a clean-up step after the Amplicon PCR. If off target products are observed in the PCR 1 products, a size selection clean up step may be required prior to proceeding with the subsequent Index PCR.

Index PCR This step adds unique dual indexes and completes the sequencing adapters using the Illumina DNA/RNA Unique Dual Index primers.

Reagents:

• Amplicon PCR Product

• Illumina DNA/RNA UD index primers

• 2x KAPA HiFi HotStart ReadyMix

• Nuclease-free water

Protocol:

Set up the following reaction in a PCR plate.

Reagent	Volume
Amplicon PCR product	5 µl
Illumina DNA/RNA UD Index Primers	10 µl
2x KAPA HiFi HotStart ReadyMix	25 µl
Water	10 µl
Total	50 µl

Pipette reactions to mix well.

Seal plate and perform PCR in a thermal cycler using the following program.

Preheat lid to 100°C. 95°C for 3 minutes 8 cycles of: — 95°C for 30 seconds — 55°C for 30 seconds — 72°C for 30 seconds 72°C for 5 minutes Hold at 4°C

Final Library Clean Up This step uses Illumina Purification beads to clean up the final library before quantification.

Required reagents:

• Illumina Purification Beads

• Freshly prepared 80% EtOH from absolute EtOH stock

• 10 mM Tris pH 8.5, nuclease-free water, or Resuspension Buffer (RSB)

Protocol

1. Centrifuge the Index PCR plate at 280 × g for 1 minute to collect condensation.

2. Vortex the Illumina Purification beads for 30 seconds to make sure that the beads are evenly dispersed.

3. Add 40 µl of Illumina Purification beads to each sample in the plate.

4. Pipette mix 10 times.

5. Incubate at room temperature for 15 minutes.

6. Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.

7. With the Index PCR plate on the magnetic stand, use a multichannel pipette to remove and discard the supernatant. Change tips between samples.

8. With the Index PCR plate on the magnetic stand, wash the beads with freshly prepared 80% ethanol as follows:

   1. Using a multichannel pipette, add 200 µl of freshly prepared 80% ethanol to each sample well.

   2. Incubate the plate on the magnetic stand for 30 seconds.

   3. Carefully remove and discard the supernatant.

9. With the Index PCR plate on the magnetic stand, perform a second ethanol wash as follows:

   1. Using a multichannel pipette, add 200 µl of freshly prepared 80% ethanol to each sample well.

   2. Incubate the plate on the magnetic stand for 30 seconds.

   3. Carefully remove and discard the supernatant.

   4. Use a P20 multichannel pipette with fine pipette tips to remove excess ethanol.

10. With the Index PCR plate still on the magnetic stand, allow the beads to air‐dry until all the EtOH is evaporated and the beads are a lighter brown color and no longer glossy.

11. Remove the Index PCR plate from the magnetic stand.

12. Using a multichannel pipette, add 27.5 µl of Resuspension Buffer (RSB), nuclease-free water, or 10 mM Tris pH 8.5 to each well of the Index PCR plate.

13. Gently pipette mix up and down 10 times or until beads are fully resuspended.

14. Incubate at room temperature for 2 minutes.

15. Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.

16. Carefully transfer 25 µl of the supernatant from each well of the Index PCR plate to a new 96‐well PCR plate. Seal well.

Validate Library Run the final libraries on a Bioanalyzer, TapeStation, Fragment Analyzer, or equivalent. Note: If non-specific bands are observed in the final libraries, additional bead or gel-based size selection and purification may be required.

The following traces were run on the TapeStation with a D5000 ScreenTape.

Example V1-V5 final library

Example V1-V6 final library

Example V1-V9 final library

Normalization and pooling Quantify library with a fluorometric quantification method specific to double stranded DNA. See Best practices for manually normalizing library concentrations for subsequent steps.

Sequencing

For V1-V5 and V1-V6 libraries:

Spike in at least 20% of the PhiX Indexed Control (1000 cycles), catalog 20151542, library to add base diversity.

• Note: this is different from the Illumina PhiX V3 control.

Loading concentration can be optimized via titration experiments. Illumina has tested loading concentrations between 120 pM and 180 pM with this library type. Each lab must validate the optimal loading concentration to use based on specific amplicon target(s) and lab workflow. See the Maximizing performance on the MiSeq™ i100 Series tech note for additional information.

Public data for sequencing and data analysis are available below.

MiSeq i100: 16S Metagenomics 2x500 reads V1-V5

• The link to run is here.

• The link to project is here.

Note: BaseSpace login is required to access BaseSpace public data.

For V1-V9 libraries: Illumina has performed limited sequencing testing for this target, and no sequencing guidance is available at this time. Labs can test sequencing this target if desired. Illumina cannot guarantee performance.

Data Analysis Illumina recommends using the DRAGEN 16S Plus analysis workflow (available in BaseSpace and onboard).

• If using a third party analysis workflow, note that for the V1-V6 and V1-V9 amplicons, the insert reads do not overlap, so reads cannot be stitched/merged. An analysis workflow that does not require insert read overlap must be used for sequencing datasets targeting these amplicons.

Supporting information The following are the reagents used for this workflow.

Consumable	Supplier
Illumina Purification Beads 100 ml or 400 ml	Illumina catalog # 20060057 or 20060058
Ethanol 200 proof (absolute) for molecular biology (500 ml)	General lab supplier
Amplicon PCR Forward Primer (Standard desalting)	General oligo synthesis supplier
Amplicon PCR Reverse Primer (Standard desalting)	General oligo synthesis supplier
2X NEB Phusion High Fidelity PCR Master Mix with HF Buffer	New England Biolabs, part # M0531
KAPA HiFi HotStart ReadyMix (2X)	KAPA Biosystems, part # KK2601
Illumina DNA/RNA UD Indexes, Tagmentation	Illumina catalog # 20091654, 20091656, 20091658, 20091660
PhiX Indexed Control (1000 cycles)	Illumina catalog # 20151542
Tris‐HCl 10 mM, pH 8.5	General lab supplier
Nuclease-free water	General lab supplier
DMSO (molecular biology grade)	General lab supplier

For any feedback or questions regarding this article (Illumina Knowledge Article #10152), contact Illumina Technical Support [email protected].