The development of chain termination sequencing, known as Sanger sequencing, forever changed biological research, allowing researchers to determine first the sequence of a gene, and ultimately of entire genomes. Since the inception of next-generation sequencing (NGS) platforms, DNA sequencing has rapidly evolved. NGS refers to various high-throughput sequencing technologies that use a wide variety of sequencing chemistries. These technologies can be categorized by the sequencing read length into short-read sequencing and long-read sequencing. Short-read sequencing technologies include: sequencing by synthesis, ion semiconductor, sequencing by ligation, and pyrosequencing. In contrast, NGS technologies that produce long sequencing reads is composed of: single-molecule real-time sequencing and nanopore sequencing.
All NGS platforms, regardless of type, depend upon the use of high-quality NGS libraries to ensure successful sequencing. A typical NGS library is composed of DNA sheared to an average uniform size that has undergone further enzymatic treatment specific to the library preparation kit and the sequencing platform. Traditional methods used to assess the quality of NGS libraries, such as agarose gel electrophoresis, lack the necessary accuracy, precision, and sensitivity to produce truly reliable results. Quality control (QC) instruments for NGS libraries must also deal with varied and challenging requirements. Numerous types of NGS libraries are available for different sequencing platforms. While each of these libraries is unique, they face similar analytic challenges, including determining fragment size (large or small), library concentration, and limited library volume.
Regardless of the NGS technology used, all require a reliable nucleic acid QC process to help ensure accurate and reproducible results. Agilent has compiled several instruments and various reagent kits into an extended Automated Electrophoresis portfolio, designed to meet the evolving demands of NGS. While these instruments, including the Bioanalyzer, Fragment Analyzer, TapeStation, and Femto Pulse systems, are each designed for the QC of nucleic acids, they each offer different advantages in terms of throughput, sensitivity, speed, and resolution. Whether the demands originate from the NGS platform or from the sample type, Agilent has the QC solution.
Successful sequencing demands optimal loading of flow cells, in turn requiring accurate and precise determination of average library size and concentration for molarity calculations. An Agilent instrument helps ensure successful sequencing with confident quantification, qualification, and sizing of short-read NGS library smears.
Accurately quantifying, qualifying, and sizing these large DNA fragments poses a significant challenge, particularly for legacy methods that are either time consuming or unable to reliably analyze large DNA fragments. An Agilent instrument provides solutions for reliable and efficient analysis of large DNA fragments to help ensure NGS success.
Several instrument offerings from Agilent provide effective solutions for the QC analysis of DNA samples. With the relevant reagent kits, the instruments provide unmatched sizing and quantification capabilities.
Short-read sequencing platforms, as the name suggests, use NGS libraries with a small average size, generally between 150 to 600 bp. Short-read sequencing provides deep coverage and has been utilized in the de novo assembly of the genomes of countless organisms. Successful sequencing demands optimal loading of flow cells, in turn requiring accurate and precise determination of average library size and concentration for molarity calculations.
The Agilent Automated Electrophoresis instruments help ensure successful sequencing with confident quantification, qualification, and sizing of short-read NGS library smears. For example:
The Bioanalyzer system is compatible with several kits covering different sizing ranges from 25 to 12,000 bp.
The Fragment Analyzer systems offer many kits, three of which are specific for short-read libraries with a sizing range from 1 to 1,500 or 6,000 bp.
The TapeStation systems provide fast run times with sizing up to 1,000 bp or 5,000 bp depending on the assay.
The Femto Pulse system is ideal for situations where the short-read NGS library concentration is low or sample conservation is crucial.
2100 Bioanalyzer system
A short-read sequencing library analyzed on a 2100 Bioanalyzer system with a High Sensitivity DNA kit.
Fragment Analyzer system
Short-read sequencing library separated using the HS NGS Fragment on a Fragment Analyzer system equipped with a short capillary array, 33 cm.
Final library of a SureSelectXT workflow analyzed with the D1000 ScreenTape assay using a TapeStation system.
Femto Pulse system
A short-read library diluted to 5 pg/ul and analyzed on the Femto Pulse system with the Ultra Sensitivity NGS kit.
Short-read sequencing provides many sub-applications to address challenging sample types and unique questions. Different types of short-read sequencing libraries can enable identification of epigenetic regions, diminish bias in sequencing libraries with PCR-free libraries, and deepen understanding of gene expression.
Chromatin Immunoprecipitation sequencing (ChIP-seq) provides crucial information on protein-DNA interactions, applying NGS to regions of genomic DNA involved with protein binding. Typical ChIP-seq workflows are resource-intensive, requiring high concentration samples for crucial QC checkpoints during library construction, limiting the application of ChIP-seq to abundant cell types.
The sensitivity of the Automated Electrophoresis instruments alleviates this restriction, dramatically decreasing the amount of sample required for quality control analysis. Successfully quantify ChIP-seq libraries with:
Final library of a ChIP-Seq workflow analyzed on a TapeStation system with the D1000 ScreenTape assay. Successful libraries show a narrow library distribution with a peak size of 120–130 bp larger than the starting material. In this example, the final library shows a maximum peak size of 318 bp, correlating to an insert size of 200 bp.
Many NGS library construction protocols require at least one PCR amplification step to ensure that there is enough material to construct a library at a sufficient concentration. While valid, amplification steps frequently introduce bias and error into the sequencing libraries. PCR-free libraries eliminate amplification steps, reducing error and removing bias from NGS library construction. Problematically, PCR-free libraries are often at such a low concentration that legacy methods of NGS library QC are unable to size, quantify, and qualify the libraries.
The Femto Pulse system provides the necessary sensitivity for the evaluation of PCR-free libraries. The Ultra Sensitivity NGS kit enables the fast and accurate QC analysis of PCR-free libraries, providing researchers with the results they need when they need them.
Small RNA Libraries
Small RNAs are involved in many important biological processes, notably gene expression. Small RNA sequencing provides researchers with further insight into these processes. Sequencing libraries made from small RNA present novel QC challenges due to the small smear size and narrow smear range.
The precision resolution of the 2100 Bioanalyzer and the Fragment Analyzer systems alleviate issues with the accurate analysis of small RNA NGS libraries. In the example shown below, with the HS Small Fragment kit for the Fragment Analyzer systems, researchers can accurately size, quantify, and qualify small RNA NGS libraries with ease.
Fragment Analyzer system
Small RNA library separated using the HS Small Fragment kit. Capillary electrophoresis performed on a Fragment Analyzer equipped with a short capillary array, 33 cm.
Recommended assays for short-read sequencing libraries
For Research Use Only. Not for use in diagnostic procedures.
Long-read sequencing libraries are composed of fragments with a large average size, extending as far as 20 to 40 kb. Sequencing long fragments of DNA provides several advantages, notably: deeper insight into genome architecture and sequencing through homopolymer runs with ease. Accurately quantifying, qualifying, and sizing these large DNA fragments poses a significant challenge, particularly for legacy methods that are either time consuming or unable to reliably analyze large DNA fragments.
Several QC solutions are available for reliable and efficient analysis of large DNA fragments, including both input genomic DNA and long-read sequencing final libraries, to help ensure NGS success.
For the Femto Pulse system, the Genomic DNA 165 kb kit (p/n FP-1002) provides optimized pulsed-field separation methods for enhanced resolution of samples up 165 kb.
Two reagent kits for the Fragment Analyzer systems are available: the Large Fragment kit (p/n DNF-492) and the HS Large Fragment 50 kb kit (p/n DNF-464). The first kit provides reliable sizing through 20 kb, while the HS Large Fragment 50 kb kit provides accurate sizing and quantification of large DNA fragments through 60 kb.
For Research Use Only. Not for use in diagnostic procedures.
Next-Generation Sequencing (NGS) Analysis Kits
Of the various methods for nucleic acid quality control analysis, the Agilent Automated Electrophoresis instruments provide accurate and versatile analysis of DNA samples. The Bioanalyzer, Fragment Analyzer, TapeStation, and Femto Pulse systems provide variable throughput, high sensitivity detection, and broad sizing ranges for reliable nucleic acid QC in NGS workflows of any type.
The Agilent 4150 TapeStation system performs reliable sample quality control (QC) through various steps of the SureSelect XT HS target enrichment and nextgeneration sequencing (NGS) library preparation workflow on the Agilent Magnis NGS Prep system.
This application note addresses and demonstrates the utility of two automated electrophoretic platforms - TapeStation and Bioanalyzer systems - at various QC check-points throughout the Agilent NGS product portfolio
This Application Note describes quality control (QC) steps in various Illumina next-generation sequencing (NGS) workflows provided as a service by the German Cancer Research Center (DKFZ) Genomics and Proteomics Core Facility.