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Quick-16S Plus NGS Library Prep Kit (V3-V4, UDI)
D6421-PS1 / D6421-PS2 / D6421-PS3 / D6421-PS4 / D6421-PS5 / D6421-PS6 / D6421-PS7 / D6421-PS8
Quick-16S Plus NGS Library Prep Kit (V3-V4, UDI)
- The most streamlined NGS kit with only 30 minutes of hands-on time for 96 samples.
- 100% automation ready with only a single PCR step and without the need for normalization.
- Real-time PCR enables absolute microbial copy number quantification.
The Quick-16S Plus NGS Library Prep Kit (V3-V4, UDI) is the fastest and simplest NGS library prep targeting the V3-V4 region of the 16S rRNA gene for high-throughput sequencing. The automation-friendly protocol utilizes a single qPCR/PCR for combined targeted amplification and barcode addition using specially designed primers. After pooling by equal volume, a single clean-up of the final library is performed, rather than massive AMPure® bead-based clean-ups. Additional library quantification analysis such as TapeStation® analysis or gel electrophoresis are not necessary. With these features, the workflow dramatically reduces the hands-on time of library preparation to only 30 minutes.
|16S V3-V4 Primer Sequences (adapters not included)||341f (CCTACGGGDGGCWGCAG, CCTAYGGGGYGCWGCAG, 17 bp) and 806r (GACTACNVGGGTMTCTAATCC, 24 bp). The forward primer 341f is a mixture of the two sequences listed.|
|Amplicon Size||The final amplicon size after 1-Step PCR (targeted amplification and barcode addition) is ~606 bp.|
|Barcode Sequences||Barcode Sequences: 10 bp barcodes, Available for download here.|
|Index Primers||Unique dual index (barcodes) to label individual samples.|
|Primer Coverage||bacteria (84.2%), archaea (75.8%), and eukaryota (0.0%).|
|Required Equipment||Microcentrifuge, plate spinner (centrifuge), 96-well real-time quantitative PCR system (SYBR Green compatible) or standard PCR system, and 96-well real-time PCR plates.|
|Sample Input||Purified microbial DNA ≤100 ng, free of PCR inhibitors.|
|Sequencing Platform||Illumina MiSeq® without the need to add custom sequencing primers. Zymo Research recommends the MiSeq® Reagent Kit v3 (600-cycle). For assistance with sample sheet setup, see Appendix F.|
Q1: What samples are compatible with this kit?
Purified total DNA from any organism free from PCR inhibitors. This system has been used by Zymo Research’s Microbiomics Services team to process samples from human, water, soil, food, plants, feces, biofilms, and much more.
Q2: What is the minimum DNA input for this kit?
The system has been tested with inputs as low as 100 femtograms with no significant impact to the performance of the kit.
Q3: Why does the kit use only a single PCR amplification step?
This single PCR step combines targeted amplification of the microbial genome and the barcoding index PCR, which allows for a simple and fast workflow.
Q4: How does the kit achieve normalization?
The combination of using Equalase qPCR Premix to yield roughly equal amounts of libraries and a carefully curated barcode index list results in similar raw sequencing reads across samples.
Q5: What is the difference between the two standards included in the kit?
The ZymoBIOMICS Microbial Community DNA Standard consists of purified DNA from 8 bacteria and 2 yeasts and is useful for assessing community profile bias. The ZymoBIOMICS 16S/ITS qPCR Standard consists of a plasmid that has a single bacterial and single fungal target and is useful for quantifying copy number. Both are useful as positive controls for the library prep process.
Q6: Why is the library amplicon size not well defined in DNA fragment analysis?
Equalase amplification and the library prep design may cause some library products to not anneal well, causing a lack of a tight band. These libraries are perfectly fine because preparation for Illumina sequencing includes denaturing libraries into single strands.
Q7: What are the differences between the D6420 and D6421 versions of this kit?
The D6421 Quick-16S Plus NGS Library Prep Kit (V3-V4, UDI) kit features unique dual indexes for sample barcoding, as opposed to the combinational barcodes utilized by the original D6420 Quick-16S Plus NGS Library Prep Kit (V3-V4). Unique dual indexes provide increased precision during sample demultiplexing, and offer improved compatibility for the Illumina® NextSeq™ 2000 sequencing platform.
|D4302-5-10||ZymoBIOMICS DNase/RNase Free Water||10 ml||$30.80|
|D6305||ZymoBIOMICS Microbial Community DNA Standard||200ng||$114.40|
|D6306||ZymoBIOMICS Microbial Community DNA Standard||2000ng||$228.80|