Library Construction

Automated workflows for fast, highly reproducible library construction
DNA library construction is a critical step in the NGS workflow, since the library DNA must be of sufficient yield and quality to generate accurate sequencing data. QIAGEN's library preparation solutions ensure maximum yields of high-quality library DNA, with optimized, automated workflows that eliminate the bottlenecks often caused by other time-consuming library preparation procedures.

Innovative solutions deliver:
  • Automated library prep on the QIAcube
  • High yields and fast, optimized procedures that provide ~50% time savings
  • Unbiased amplification with an optional, high-fidelity amplification step
  • Precise size selection of DNA fragments 
  • High precision, qPCR-based library quantification
DNAseq NGS workflow
Automated library construction
Precise, spin-column-based size selection of library DNA
qPCR-based library quantification
Automated library construction with a fast and highly reproducible workflow
GeneRead Library Prep Kits provide an efficient and optimized workflow, generating high yields of DNA library with minimal sequence bias and low error rates. The complete library preparation workflow can be automated on the QIAcube, saving time while ensuring high reproducibility and standardization. DNA libraries are ready for use on NGS platforms from Illumina and Life Technologies.

Efficient ligation reactions and an optional, high-fidelity amplification step ensure superior library yields and quality from as low as 50 ng starting material (see figure High yields of DNA library with uniform coverage distribution and High yields of library DNA). The fast, one-tube procedure and optimized workflow significantly save time and effort and minimize the variability caused by handling, along with the risk of contamination (see figures Optimized workflow for Life Technologies and Optimized workflow for Illumina).

To ensure maximum yields from minimum amounts of starting material, GeneRead Library Prep Kits include an innovative, high-fidelity master mix for an optional amplification step. The unique, highly specific and processive enzyme GeneRead HiFi Polymerase, a unique provides accurate amplification of library DNA with low error rates and minimum bias (see figure Low error rates with minimal sequence bias). With standard PCR amplification procedures, regions of DNA with high AT or GC content can result in little or no amplification, leading to misleading sequence data and NGS results. GeneRead HiFi Polymerase, together with its unique buffer formulation, ensures uniform amplification of genomic regions that contain highly variable GC content, thereby ensuring even coverage in subsequent sequencing reactions (see figure Better sequence coverage and minimal bias).

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Precise size-selection of library DNA
The GeneRead Size Selection Kit uses a convenient, spin-column-based procedure to ensure precise size selection of the DNA library while effectively removing adapter dimers or monomers (see figure Precise size selection). Automatable on the QIAcube, the easy-to-use protocol eliminates tedious handling procedures and ensures there is no risk of carryover of potentially inhibitory ethanol or beads.

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qPCR-based library quantification
One of the most important factors in an NGS experiment is accurate quantification of the prepared library to ensure quality reads and efficient data generation. Underestimation of amplifiable library molecules leads to mixed signals and non-resolvable data; conversely, overestimation results in poor yield of template-carrying beads (Ion Torrent platform) or clusters (Illumina platform) and reduced use of sequencing capacity.

The GeneRead Library Quant System uses real-time PCR to quantify NGS library molecules (see figures Targeted enrichment NGS workflow and PCR-enabled target enrichment of genes of interest). It specifically quantifies DNA molecules with adaptors at both ends, which are the only amplifiable molecules during emulsion PCR (Ion Torrent platform) or bridge PCR (Illumina platform) and therefore enable highly accurate quantification of amplifiable library molecules (see figure Reliable NGS library quantification and Quantification of libraries with concentrations below the detection limit of conventional methods). The high sensitivity of real-time PCR allows quantification of library DNA with very low concentrations, even below the detection threshold of conventional spectrophotometric methods.

The GeneRead Library Quantification System quantifies DNA after targeted PCR-enabled amplification with the GeneRead DNAseq Gene Panel System to monitor the efficiency of PCR-enrichment of targeted genes of interest with integrated controls. It can also be used for the quantification of DNA for any of the following applications:
  • Whole genome
  • Exome
  • RNAseq/transcriptome
  • ChIPseq
  • Methylation
  • Targeted amplification of GOIs with any other system



Top products
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GeneRead qPCR SYBR Green Mastermix
For use with the GeneRead Library Quant System
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GeneRead DNAseq Library Quantification Kit
For qPCR-enabled quantification of NGS libraries before sequencing
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QIAseq Library Quant Array Kit
For qPCR-enabled quantification of NGS libraries
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GeneRead DNA Library I Core Kit (12), Gene Read DNA I Amp Kit (100), GeneRead Adapter I Set 1-plex (12)
For fast and efficient preparation of DNA libraries for use in NGS applications
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GeneRead Size Selection Kit (50)
For quick and reliable removal of DNA fragments <150 bp for library preparation in NGS applications
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Kit Handbooks
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For preparation of DNA libraries for next-generation sequencing (NGS) applications that use Illumina instruments
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For preparation of DNA libraries for next-generation sequencing (NGS) applications that use Ion Torrent instruments from Life Technologies
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Quick-Start Protocols
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Brochures & Guides
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Accelerate your NGS performance through Sample to Insight solutions
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Accelerate your NGS performance through Sample to Insight solutions
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Images
Better sequence coverage and minimal bias when amplifying GC- or AT-rich sequences
Better sequence coverage and minimal bias.
A mixture of highly GC-rich Bordetella pertussis gDNA (GC content 67.7%) and highly AT-rich Streptobacillus moniliformis gDNA (GC content 26.3%) was pooled, made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit, and amplified with the GeneRead Library Amp (I) Kit, which contains HiFi Polymerase, or a kit from Supplier K. The amplified libraries were sequenced using the Illumina MiSeq instrument, and fidelity and sequence coverage was analyzed using the Galaxy platform. The GeneRead Library Prep Kit provided greater sequence coverage in GC- and AT-rich areas of DNA, compared to the kit from Supplier K.
GeneRead DNAseq Gene Panel System targeted enrichment NGS workflow
Targeted enrichment NGS workflow.
First, extract DNA (the QIAamp DNA Mini Kit or QIAamp DNA FFPE Tissue Kit is recommended), and then use GeneRead DNAseq Gene Panels for targeted exon enrichment. Then construct your NGS library, quantify and quality-control using the GeneRead Library Quantification System, and perform NGS and data analysis using the QIAGEN NGS Data Analysis Web Portal.
The GeneRead Library Prep Kit delivers high yields of library DNA.
High yields of library DNA.
Genomic DNA (1 μg) from E. coli strain DH10B was sheared using a Covaris instrument and made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit or a kit from another supplier. The library was eluted in 18 μl Buffer EB and library concentrations were determined by quantitative PCR.
Low error rates with minimal sequence bias due to high-fidelity amplification.
Low error rates with minimal sequence bias.
A mixture of highly GC-rich Bordetella pertussis gDNA (GC content 67.7%) and highly AT-rich Streptobacillus moniliformis gDNA (GC content 26.3%) was pooled, made into an Illumina-compatible DNA library using the GeneRead Library (I) Core Kit, and amplified with the GeneRead Library (I) Amp Kit, which contains HiFi Polymerase, or a kit from Supplier K. The amplified libraries were sequenced using the Illumina MiSeq instrument, and fidelity and sequence coverage were analyzed using the Galaxy platform. [A] Low error rates and [B] greater cumulated sequence coverage demonstrate that the GeneRead Library (I) Amp Kit provides superior library amplification compared to the kit from Supplier K.
Optimized workflow allows more rapid and efficient preparation of Illumina-compatible DNA libraries.
Optimized workflow for Illumina.
The GeneRead Library Prep (I) Kit uses an optimized one-tube protocol, with fewer cleanup steps and optional high-fidelity library amplification. The hands-on time and total time required for preparation of library DNA is significantly reduced compared to the library preparation system from Supplier I.
Optimized workflow allows more rapid and efficient preparation of Life Technologies-compatible DNA libraries.
Optimized workflow for Life Technologies.
The GeneRead Library Prep (L) Kit uses an optimized, one-tube procedure, with fewer cleanup steps and optional high-fidelity library amplification. The hands-on time and total time required for preparation of library DNA is significantly reduced compared to the library preparation system from Supplier L.
PCR-enabled target enrichment of genes of interest (GOI)
PCR-enabled target enrichment of genes of interest.
The principle of the GeneRead DNAseq System is to employ overlapping primer sets across the exonic portions of a gene or genes to maximize target coverage and minimize nonspecific amplification.
Precise size selection
Precise size selection.
The GeneRead Size Selection Kit effectively removes adapter dimers and adapter monomers following library preparation. A scaled-up image of the above data showing the correct size distribution of Illumina-compatible library fragments following size selection is shown in inset. FU: Fluorescence units.
GeneRead Library Quant System enables quantification of libraries with concentrations below the detection limit of conventional methods.
Quantification of libraries with concentrations below the detection limit of conventional methods.
The GeneRead Library Quant Array's high sensitivity and broad dynamic range enable the quantification of both NGS-L1 and NGS-L2 libraries. By contrast, the kit from Suppler A (for use with the Agilent 2100 BioAnalyzer) quantified only the NGS-L1 library; with this kit, the NGS-L2 library concentration was too low for quantification.
Reliable NGS library quantification with minimal variability of DNA standards from lot to lot.
Reliable NGS library quantification.
The DNA standards for the Illumina MiSeq platform from three different lots were used to prepare five sequential 10-fold dilutions. The diluted DNA standards were mixed with a library-specific PCR primer assay and GeneRead qPCR SYBR Green Mastermix (from three different lots) and were subjected to real-time PCR. The minimal variation in CT values for diluted DNA standards from three different lots confirms the reliability of the Gene Read Library Quant System.
High yields of library DNA with uniform coverage distribution.
High yields of library DNA with uniform coverage distribution.
[A] Genomic DNA (50 ng) was sheared using a Covaris instrument and made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit. Sequencing using an Illumina MiSeq instrument revealed a median coverage of 49-fold with uniform coverage distribution. [B] Genomic DNA (1 µg) from E. coli strain DH10B was sheared and used to generate an Ion Torrent-compatible DNA library using the GeneRead Library Prep (L) Kit or a kit from another supplier. After amplification for 10 cycles, both libraries were sequenced on an Ion Torrent PGM instrument and the cumulated normalized coverage was analyzed.