Amplicon sequencing

Overview

• Integrated library normalization—Normalase™ technology enables a streamlined library balancing and pooling process without the need to quantify samples
  
• Customizable—add in additional targets to custom amplicon sequencing panels or predesigned panels
• Time saving—single-tube workflow, go from cDNA to normalized library pool in approximately 3 hours

What is amplicon sequencing?

Targeted DNA sequencing methods, such as amplicon sequencing, provide an opportunity for the interrogation of specific regions of the genome so that researchers can obtain information about the genetic regions they care about the most. In amplicon sequencing, highly multiplexed PCR generates amplicons of targeted regions of genomic DNA, after which adapters are added via PCR using indexing primers with either combinatorial dual indexes (CDIs) or unique dual indexes (UDIs).  The final library fragments are pooled and sequenced. Index sequences are then used to demultiplex the data bioinformatically, and the sequences of the targeted genomic regions are used for further analysis.

Applications

Amplicon sequencing benefits these areas of research:

• Identification of rare variants
• Identification of hot-spot mutations
• CRISPR genome editing confirmation
• Oncology and virology
• Whole genome sequencing confirmation
• Genotyping

This highly multiplexed PCR approach provides flexibility for a wide range of experimental designs while helping to reduce costs and workflow time. It is also particularly useful for the discovery of rare somatic mutations in complex samples (e.g., tumors mixed with germline DNA) and hard-to-sequence areas (e.g., GC-rich regions).

For an in-depth discussion on this method, see our Amplicon sequencing technical overview.

Predesigned and custom amplicon sequencing panels

IDT offers a wide variety of different predesigned panels, but if research takes you in an uncharted direction, custom amplicon sequencing panels can be created for your specific needs.

Table 1. IDT xGen Amplicon Panels.

^† The 16S Amplicon Panel v2 and xGen ITS1 Amplicon Panel, both of which are covered in this protocol, are separate products that should be purchased individually.

xGen 16S rRNA gene sequencing workflow

Analysis of sequencing data obtained using the xGen 16S Amplicon Panel v2 is presented here as an example of a research question that may be addressed using one of IDT’s predesigned panels. The xGen 16S Amplicon Panel v2 enables library construction from DNA using tiled primer pairs to target the V1–V9 variable regions of the 16S rRNA gene (Figure 1). The xGen 16S Amplicon Panel v2 facilitates the analysis of microbial communities that consist of bacteria and archaea using a single primer pool.

The xGen 16S Amplicon Panel v2, like all the IDT xGen Amplicon Panels (Table 1), utilizes multiple overlapping amplicons in a single tube, using a rapid, two-hour workflow to prepare ready-to-sequence libraries. IDT’s PCR1+PCR2 workflow generates reliable libraries, even from low input quantities (Figure 2). Then libraries can optionally be normalized enzymatically with the xGen Normalase™ technology, which is included with the xGen 16S Amplicon Panel v2. This integrated normalization step can save time and resources.

The xGen Amplicon Panels can also be customized for additional targets by the addition of other predesigned panels or a custom amplicon panel.

The sequencing data generated from xGen Amplicon libraries can be processed and analyzed using the recommendations in Primerclip-A Tool for Trimming Primer Sequences Application Note. For sequences generated with the xGen 16S rRNA v2 Amplicon Panel, the 16S SNAP APP-An automated pipeline for community analysis using multiple 16S rRNA variable regions tool can be used, further streamlining your amplicon based sequencing workflow.

Method data

Coverage of xGen Amplicon Panel Technology

To fully analyze complex microbial communities, amplicon sequencing often targets one variable region of the 16S rRNA gene. The IDT xGen Amplicon Panel for 16S rRNA analysis amplifies all nine variable regions of the 16S rRNA gene. To assess the ability of the xGen 16S Amplicon Panel v2 to capture diverse microbial communities, libraries were prepared using a commercially available standard (MSA-1003, ATCC; n = 24) and the xGen 16S Amplicon Panel v2 (covering the V1–V9 regions of the 16S rRNA gene); a panel targeting only the V3–V3 16S rRNA gene regions; and IDT’s legacy Accel-Amplicon 16S+ITS panel. The resulting libraries were sequenced on a MiSeq^® (Illumina^®) instrument (2 x 150 PE) without the use of the phiX reagent.

Libraries were also prepared using 10–50 ng of DNA from swine manure (n = 2) and 10 pg and 1 ng of MSA-1003 to establish the flexibility of the sample input into the xGen 16S Amplicon Panel v2. Amplicons were generated using xGen 16S Amplicon Panel v2 protocol and listed cycling conditions, as well as the legacy Accel-Amplicon 16S+ITS panel. Amplicon libraries were sequenced using both 2 x 150 and 2 x 300 PE sequencing chemistries on the MiSeq^® platform (Illumina).

The resulting sequences were analyzed using recommendations found in the 16S SNAP APP-An automated pipeline for community analysis using multiple 16S rRNA variable regions. 

The resulting sequences from this workflow clearly illustrate the ability of the xGen 16S Amplicon Panel v2 to capture the diversity microbial communities from different sample types, inputs, and using different sequencing chemistries (Figures 1 and 2). This fast, efficient, and flexible workflow provides researchers with all the necessary reagents to produce reliable 16S rRNA gene sequencing datasets across all nine variable regions of the gene.

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