16S Microbiome + ITS NGS Assay
The Viennalab 16S Microbiome + ITS NGS Assay is a library preparation kit for characterising the human microbiome by next-generation sequencing. It targets the variable V3-V4 regions of the bacterial 16S rRNA gene and, optionally, the fungal ITS2 region. Research use only. The kit supplies two primer pools, MIB1 for V3-V4 and FUN1 for ITS2, and dual-index primers for up to 96 unique index combinations. One kit covers 96 library preparations.
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This kit in detail
- Bacterial 16S V3-V4 and, optionally, fungal ITS2
- Two primer pools: MIB1 – V3V4 Mix and FUN1 – ITS2 Mix
- Two-stage PCR with dual indexing, 96 index combinations per kit
- DNA input 2.5 to 25 ng for 16S alone, 25 to 50 ng for 16S plus ITS
- Illumina MiSeq with at least 2×250 bp reads
- 96 library preparations per kit, REF 9-131, 9-132 and 9-133
The Viennalab 16S Microbiome + ITS NGS Assay prepares amplicon libraries for the bacterial and fungal characterisation of the human microbiome. Research use only.
Regions targeted
The MIB1 – V3V4 Mix amplifies the highly variable V3-V4 regions of the bacterial 16S rRNA gene, giving an amplicon of around 600 bp. The FUN1 – ITS2 Mix amplifies the fungal nuclear ribosomal ITS2 region, 440 to 540 bp. The fungal part is optional: the assay can be run 16S only, and the required DNA input differs between the two modes.
Method and requirements
Library preparation is a two-stage PCR with dual indexing. The DNA input is 2.5 to 25 ng (0.5 to 5 ng/µl) for 16S alone and 25 to 50 ng (5 to 10 ng/µl) for 16S plus ITS2. Sequencing runs on the Illumina MiSeq and needs at least 2×250 bp reads, with a minimum of 50,000 reads per sample recommended. Analysis is supported by the ViennaLab MicrobeCalc tool and the Microbiome Analysis Webtool.
Index sets and pooling
Three index sets exist, REF 9-131 (Set A), 9-132 (Set B) and 9-133 (Set C), each with 96 reactions. A single set covers 96 index combinations; combining the three allows up to 288 samples to be pooled in one run. A 16-reaction pack is available as REF 9-131-16.
Not supplied
DNA extraction reagents are not supplied. For stool the manufacturer recommends the Norgen Biotek Stool DNA Isolation Kit. No positive control, negative control or microbial community standard is supplied.
Regulatory status
This kit is for research use only.
[REF 9-131] – Set A
- Activation code sticker for 100 analyses
- Master Mix
- PCR1 V3–V4 Primer Mix (bacterial)
- PCR1 ITS2 Primer Mix (fungal, optional)
- PCR2 Indexing Forward Primers Set A (F1–F8)
- PCR2 Indexing Reverse Primers (R1–R12)
- Magnetic Beads
[REF 9-132] – Set B
- Activation code sticker for 100 analyses
- Master Mix
- PCR1 V3–V4 Primer Mix (bacterial)
- PCR1 ITS2 Primer Mix (fungal, optional)
- PCR2 Indexing Forward Primers Set B (F9–F16)
- PCR2 Indexing Reverse Primers (R1–R12)
- Magnetic Beads
[REF 9-133] – Set C
- Activation code sticker for 100 analyses
- Master Mix
- PCR1 V3–V4 Primer Mix (bacterial)
- PCR1 ITS2 Primer Mix (fungal, optional)
- PCR2 Indexing Forward Primers Set C (F17–F24)
- PCR2 Indexing Reverse Primers (R1–R12)
- Magnetic Beads
Note: REF 9-131, 9-132, and 9-133 differ only in their indexing primer sets. For sequencing more than 96 samples on a single flow cell, order different sets (e.g., Set A and Set B) to ensure all libraries have unique index combinations.
[REF 9-131-16] – Set A (16 Reactions)
- Activation code sticker for 16 analyses
- Master Mix
- PCR1 V3–V4 Primer Mix (bacterial)
- PCR1 ITS2 Primer Mix (fungal, optional)
- PCR2 Indexing Primers (F1–F4 and R1–R4)
- Magnetic Beads
Note: Indexing primers in REF 9-131-16 overlap with Set A (REF 9-131).
Sequencing Services: A service for the sequencing of the 16S rRNA gene libraries on a MiSeq or NextSeq instrument, with various read lengths and options available.
The kit also includes instructions for use, as well as a protocol for the preparation and sequencing of 16S rRNA gene libraries using the provided reagents and services.
Datasheets and regulatory documentation
Everything your quality team needs for evaluation and procurement.
Instructions for Use
This kit, put to work
Protocol detail and workflow guidance from labs already running this kit, straight from our application notes library.
All application notesFrequently paired with this kit
Other assays and instruments lab teams add alongside the 16S Microbiome + ITS NGS Assay.
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