QIAseq FX DNA Library Kit

Fast and efficient enzymatic library conversion

The new QIAseq FX DNA Library Kit takes you from20 pg – 1 μg genomic DNA to sequencer-ready, amplified libraries in just2.5 hours. It’s faster and easier to automate than mechanicalshearing but still generates the high-quality libraries you need for whole genome sequencing ofany organism (see figure: Workflow). The workflow reliably generates reproducible DNA fragments of customizable sizes. (see figures: Customizable fragment sizes and High Reproducibility)

Low GC bias for superior library quality

TheQIAseq FX DNA Library Kit performs library preparations with minimal bias by using sequence-independent enzymes for fragmentation – delivering results comparable to mechanical shearing. (see figure: Minimal GC bias compared to other enzymatic methods)

Superior coverage distribution and lower duplication rate

QIAseq FX technology with its minimal sequence bias ensures that the majority of genomic targets have very similar total coveragedepth. This reduces the need for additional sequencing to bring low-coverage targets upto an interpretable coverage range, saving time and resources.Sequencing libraries generated by the QIAseq FX method have high complexity and low duplicate rates, overcoming the shortcoming commonly associated with other enzymatic fragmentation methods. (see figures:Superior coverage distribution and Lower duplication rate)

PCR-free libraries from as little as 100 ng input

Fully functional dual bar-coded adapters and optional high-fidelity library amplification reagents enable the use of QIAseq FX fragmentation and adapter ligation chemistries to generate PCR-free libraries in under two hours total workflow time. Without additional PCR library amplification, the QIAseq FX DNA Library Kit can consistently generate the higher than 2 nM PCR-free library concentration needed for sequencing on an Illumina MiSeq or NextSeq 500 – from as little as 100 ng input DNA. (see figure: No significant differences between coverage of high or low GC genomic regions)

QIAGEN’s QIAseq FX technology incorporates enzymatic DNA fragmentation into a streamlined, optimized protocol that combines fragmentation and adapter ligation in one reaction, saving time and preventing errors. The QIAseq FX kit contains a novel nuclease formulation that digests dsDNA in a random fashion without sequence preference, generating sequencing libraries with minimal bias. The fragment size is tunable simply by changing incubation time of the dsDNA with the nuclease, enabling different sequencing applications.Optimized enzyme and buffer compositions ensure high sequencing library yield. Streamlined library construction protocols also enable straightforward automation of library prep on different liquid-handling platforms.

The QIAGEN QIAseq FX DNA Library Kit provides a fast, fully enzymatic procedure, from DNA fragmentation to NGS library, with no cleanup steps until after adapters have been ligated to the sample DNA.

Overview

The QIAseq FX DNA Library Kit consists of three, easy-to-follow steps starting from genomic DNA and ending with sequencer-ready NGS libraries. Beginning with combined DNA fragmentation and end modification, sample fragments are prepared allowing for the binding of dual bar-coded adapters. For samples that are less than 100 ng of input DNA or if large amounts of library DNA are required in later applications, an optional DNA amplification step can be performed with reagents conveniently provided in this kit.

DNA fragmentation and adapter ligation

Samples consisting of longer DNA fragments, such as genomic DNA or amplicons from long-range PCR, are first enzymatically sheared into smaller fragments. The median fragment size is dependent on the applications and sequencing read length, and can be adjusted by varying the DNA fragmentation reaction conditions. The fragmented DNA is directly end-repaired, and an "A’ is added to the 3’ ends during the FX reaction ­– making the DNA fragments ready for adapter ligation. Following this step, platform-specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding dual bar-coded libraries to a flow cell for sequencing, allowing for PCR amplification of adapter-ligated fragments and for binding of standard Illumina sequencing primers.

DNA amplification

To ensure maximum yields from limited amounts of starting material, a high-fidelity amplification step can be performed using the reagents included in the QIAseq FX DNA Library Kit. The proprietary HiFi PCR Master Mix can evenly amplify DNA regions with vastly different GC contents, minimizing sequencing bias caused by PCR.

Library construction

Dual bar-coded, plate-format adapters are included with the QIAseq FX DNA Library Kit. Each well contains a single-use adapter consisting of a unique combination of two eight-nucleotide identification bar codes. By combining one D5 bar code and one D7 bar code in each ready-to-use adapter, QIAseq FX kits support up to 24-plex or 96-plex pooling prior to sequencing.

Cleanup and removal of adapter-dimers

Following library construction, the reaction cleanup and removal of adapter dimers can be achieved by using Agencourt^® AMPure^® XP Beads, enabling easy automation on various high throughput automation platforms.