Amplicon sequencing is a method for preparing focused NGS libraries from gDNA using PCR reactions.
Single amplicons
A first PCR process utilizes custom PCR primers to generate the amplicon that will be the focus of the sequencing designed. The primers amplify your specific region of interest plus an additional 20 bp region upstream of each primer, called Linkers. We use the Linkers SP1 for the forward primer and SP2 for the reverse primer.
SP1: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT- [locus specific sequence – Forward primer]
SP2: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT- [locus specific sequence – Reverse Primer]
The first step PCR can be performed in your lab or at Syntezza. You can order the primers from IDT, perform the first stage PCR at your lab and our staff will pick-up your PCR products and bring them to our laboratory in Jerusalem. We strongly recommend ordering primers produced using IDT’s ultramers chemistry.
We will perform a second PCR process to attach barcodes to each sample and, following QC steps, perform the NGS sequencing and any required bioinformatic analysis of the results. Pricing for this second step PCR is available from ₪45 to ₪60 per sample.
Alternatively, you can email your custom primer sequences to NGS@syntezza.com. We will order the primers with the added linker sequences and perform the first step PCR as well as the second step PCR to finalize your library in preparation for NGS sequencing. Price for the complete Amplicon-Seq library preparation from gDNA is available for the cost of the custom primers plus ₪75 – ₪90 per sample.
Metagenomics 16-S Panel
A better approach to 16-S Amplicon-Seq, we use the IDT xGen 16S Amplicon Panel v2 that incorporates a pool of tiled multiplex primer pairs targeting each of the nine V1-V9 variable rRNA regions. This method allows NGS analysis of complex microbial communities (e.g., bacteria, archaea, fungi), providing superior representation of each genus of the diverse microbial communities.
Custom Targeted Amplicon Panels (up to 500 targets)
These primer pools are custom-designed to create multiple overlapping amplicons for specific genetic targets. Learn More
Pre-designed Targeted Amplicon Panels
>We work with IDT’s pre-designed amplicon panels.
Amplicon-Seq for CRISPR
The rhAmpSeq CRISPR Analysis System from IDT allows quick and accurate quantification of CRISPR-Cas edits. The system uses a proprietary RNAse H2-dependent PCR technology to generate amplicon libraries for targeted sequencing and includes an advanced but accessible cloud-based data analysis pipeline for quantification of on- and off-target edits.Learn More