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Amplicon sequencing

Single primer pool gives flexibility to get sufficient sequencing depth and coverage of all 9 variable regions of 16S rRNA.

xGen™ NGS—made for amplicon sequencing.

Overview

  • Near complete coverage—cover all 9 variable regions of the 16S rRNA gene available as a single primer pool
  • Integrated library normalization—enables streamlined library balancing and pooling process without the need to quantify samples
  • xGen 16S amplicon chemistry—generates diverse clusters without PhiX or phased primers, recovering >20% of reads
  • Customizable—add in additional targets including antibiotic resistance or virulence genes
  • Time saving—Single tube, go from cDNA-to-normalized- library-pool in 3 hours

Amplicon sequencing

Targeted DNA sequencing methods allow for the interrogation of specific regions of the genome so that researchers can obtain information about the genetic regions they care about the most. One commonly employed targeted DNA sequencing approach is amplicon-based sequencing. Amplicons are DNA products generated by PCR, pooled, and then sequenced. Typically, this method employs multiplexed primer pairs designed to amplify multiple regions of interest followed by NGS (next generation sequencing).

This method allows for multiplexing of samples where hundreds of amplicons can be determined simultaneously, therefore it can effectively cover large genomic regions.

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 ultra-high multiplexed PCR approach allows 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 (i.e., GC-rich regions).

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

xGen 16S rRNA gene sequencing workflow

The xGen 16S Amplicon Panel v2, enables library construction from DNA using tiled primer pairs to target the V1-V9 variable rRNA gene regions (Figure 1). The xGen 16S Amplicon Panel v2 facilitates the analysis of complex microbial communities (e.g., bacteria, archaea) using a single primer pool.

The xGen 16S Amplicon Panel v2 utilizes multiple overlapping amplicons in a single tube, using a rapid, 2-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 including antibiotic resistance or virulence genes, allowing sub-genera level identification and functional analysis. Further, IDT offers the predesigned xGen ITS1 Amplicon Panel for the characterization of fungi.

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

Amplicon Workflow

Icons Library_White Outline_85x85_Extraction

Extraction

Icons Library_White Outline_85x85_Sequence and analyze

Sequencing & analysis

IDT ALIGN Program

Method data

Coverage of all variable regions of the 16S rRNA gene

To assess the ability 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 swine manure (n = 2) and 10 pg and 1 ng samples of MSA-1003 to establish the flexibility of sample input into the xGen 16S Amplicon Panel v2. Amplicons were generated using xGen 16S Amplicon Panel v2 protocol and listed cycling conditions, and 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.

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 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.

Figure 1. The xGen 16S Amplicon Panel v2 provides more complete representation of diverse microbial communities. The xGen 16S Amplicon Panel v2 panel covering V1–V9 regions of 16S rRNA provides accurate representation of each genus in a commercially available standard (MSA-1003) compared to libraries interrogating the V3–V4 region alone. The legacy Accel-Amplicon 16S+ITS panel and the new xGen 16S Amplicon Panel v2 show similar relative abundance results. Strains were present at levels from 0.02% to 18% in MSA-1003. Boxed organisms were not detected with sole use of the V3–V4 region. Libraries were sequenced on a MiSeq® (Illumina®) instrument without the use of the PhiX reagent (n = 1, representative dataset). As shown in the examples in this figure, by not losing reads to PhiX or having to sequence deeper due to the use of phased primers, an increased number of samples can be fit on a sequencing run, thereby increasing sequencing efficiency, while still achieving quality data.

Figure 2. Consistent function with varying biomass, sample type, and read length. Using the same protocol and cycling conditions, input quantities of 10–V9 regions of 16S rRNA provides accurate representation of each genus in a commercially available standard (50 ng of swine manure (top left) identified the same quantities and types of genera. In addition, the 10 pg sample of MSA-1003 (bottom left) had the same relative abundance and type of genera as the 1 ng sample. The legacy Accel-Amplicon 16S+ITS panel and the new xGen 16S Amplicon Panel v2 products gave similar results when comparing the 2 x 150 and 2 x 300 PE sequencing read percentages; a comparable number of genera were identified from swine manure samples (right table).

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*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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