Fast Library Prep Protocol
There are two main steps involved in 16S V1-V3 amplicon processing: an initial PCR amplification using customized PCR primers that target the V1-V3 domains, and a subsequent PCR amplification that integrates relevant flow cell binding domains and unique 12 base pair sample indices. The limited number of cleanup steps ensures maximum recovery of amplicons for downstream sequencing.
Optimized Protocol Offers Lower PCR Bias and Fewer Off-target Reads
The protocol incorporated in the NEXTFLEX 16S V1 – V3 Amplicon-Seq Kit offers better sequencing results than can be obtained using traditional 16S sequencing protocols. The incorporation of the second PCR step in the protocol for the addition of the sample-specific index reduces the number of off-target reads typically encountered during amplicon sequencing.
Automate your 16S V1 – V3 Library Prep to Increase Throughput and Reduce Errors
A NEXTFLEX® automation protocol is now available on the Sciclone® NGS and NGSx workstations to help labs increase their throughput and reduce human errors. Automated versions of the NEXTFLEX® 16S V1 – V3 Amplicon-Seq Library Prep kits are available by request. Please inquire for additional information at NGS@perkinelmer.com. Automation kits are custom items and require a minimum of 2 weeks lead time.
KIT CONTENTS
- NEXTFLEX® PCR Master Mix
- NEXTFLEX® 16S V1-V3 PCR I Primer Mix
- NEXTFLEX® PCR II Barcoded Primer Mix
- Resuspension Buffer
- Nuclease-free Water
REQUIRED MATERIALS NOT PROVIDED
- 1 ng – 50 ng high-quality genomic DNA in up to 36 µL nuclease-free water for each library
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- Adhesive PCR Plate Seal (Bio-Rad®, Cat # MSB1001)
- Agencourt® AMPure® XP 5 mL (Beckman Coulter® Genomics, Cat # A63880)
- Magnetic Stand – 96 (Thermo Fisher® Scientific, Cat # AM10027) or similar
- Thermocycler
- 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
- Vortex
- 80% Ethanol, freshly prepared (room temperature)
SEQUENCES
- Sequences of NEXTFLEX 16S V1-V3 Amplicon-Seq Barcoded Primers Indexes – Excel / PDF
APPLICATION NOTE
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