QIAseq 1-Step Amplicon Library Kit

Save time with targeted resequencing and amplicon sequencing applications by taking advantage of the fastest library prep solution on the market – the QIAseq 1-Step Amplicon Library Kit. By combining end-repair and ligation, this new kit offers a fast and efficient 30-minute procedure allowing you to prepare high-quality, artifact-free libraries from any QIAGEN GeneRead Targeted DNA Panel and Ampli-Seq, Tru-Seq or PCR amplicons. The resulting NGS library can be used on any Illumina NGS instrument.

Significant time savings – from amplicon to high-quality, NGS-ready library in just 30 minutes

Traditional NGS library prep can be laborious, taking anything from 2 to 3 hours to accomplish. The QIAseq 1-Step Amplicon Library Kit offers a faster, more efficient alternative, allowing reliable NGS library prep from pools of PCR fragments from gene panels or multiplexed PCR. Utilizing a novel, combined end-repair/ligation reaction, the kit streamlines the entire NGS library preparation process to just 30 minutes (see figures  Optimized One-step Library Construction and  Overview of a Complete Targeted Resequencing NGS Workflow Including the QIAseq 1-Step Amplicon Library Kit). It incorporates a one-tube reaction that saves you time, allowing you to focus on sequencing and data analysis. The entire procedure can be performed at room temperature, enabling automation on instruments lacking a thermo-block. The one-tube protocol eliminates the need for transferring reagents, increasing efficiency and more effectively capturing insert amplicons, while also reducing the risk of contamination or sample mix-up, which can occur with manual processing. The procedure is optionally PCR-free to avoid introducing sequence duplicates or PCR-bias, and generates high-quality libraries optimized for sequencing on any Illumina sequencing instrument from a range of input materials.

Excellent sequencing metrics

Typical libraries have excellent sequencing metrics, minimal adapter-adapter ligation product, and specificity and accurately reflect the evenness and sensitivity of the input products. (see figure  Typical Results).

Purified amplicons from gene panels or multiplex PCR are converted to Illumina-compatible NGS libraries using a single, enzymatic library construction step. During this reaction, amplicons are simultaneously prepared for ligation and barcoded adapters are ligated to both ends of the DNA inserts. The adapters contain sequences required for the PCR enrichment of the subsequent library, for flow-cell-binding during bridge amplification and for sequencing primer binding sites for paired-end and multiplexed sequencing.
Following library construction, excess adapters, adapter dimers and other reaction components are removed via precipitation onto Agencourt AMPure XP beads. This procedure is carried out at room temperature, and can be easily automated on various liquid-handling platforms for high-throughput applications. Following library purification, a high-fidelity library enrichment step can be performed to generate sufficient library from low amounts of starting material. This reaction relies on a high-fidelity DNA polymerase and optimized buffer conditions that ensure minimum GC bias and extremely low error rates.
This kit is compatible with:
• PCR products generated with the GeneRead v2 DNAseq Targeted Panels
• PCR products generated with other custom or commercial gene panels
• PCR products generated with the QIAGEN Multiplex PCR Kit or other QIAGEN PCR reagents
• Multiplexed PCR amplicons generated with Taq or Taq derivatives
The novel one-step reaction requires PCR amplicons to contain 3’ A-overhangs for efficient ligation. Taq polymerase, the most commonly used thermostable DNA polymerase, and its derivatives, by default carry out this non-templated A-addition during the PCR reaction. Taq and Taq-derivatives have attributes that make them amenable to multiplex PCR, and many commercial gene panels employ a Taq-based enzyme. In contrast to Taq, other polymerases with strong 3’–5’ exonuclease activities do not carry out this reaction. While these enzymes are not commonly used for multiplexed PCR, amplicons produced with such enzymes are still compatible with the QIAseq 1-Step Amplicon Library Kit, but require A-tailing prior to ligation. Please consult the handbook for details.
Compatible sequencing platforms:
• Illumina HiSeq
• Illumina MiSeq
• Illumina NextSeq
• Illumina MiniSeq