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Next-Generation Sequencing Library Preparation

NGS Analysis with the Agilent Automated Electrophoresis portfolio

What is Next-Generation Sequencing (NGS)?

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.

Short-Read Sequencing

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.

Long-Read Sequencing

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.

Analysis Kits

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.

Application Notes

Don't stop learning here, read more about NGS analysis in our application notes.

Short-Read Sequencing

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.

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

Short-read sequencing library separated using the HS NGS Fragment on a Fragment Analyzer system equipped with a short capillary array, 33 cm.

TapeStation system

SureSelectXT workflow analyzed with the D1000 ScreenTape assay using a TapeStation system

Final library of a SureSelectXT workflow analyzed with the D1000 ScreenTape assay using a TapeStation system.

Femto Pulse system

short-read library analyzed on the Femto Pulse system with the Ultra Sensitivity NGS kit

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.

ChiP-Seq

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:

TapeStation system

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.

ChiP-Seq workflow on the TapeStation

PCR-Free Libraries

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.

Small RNA library separated using the HS Small Fragment kit

Recommended assays for short-read sequencing libraries

2100 Bioanalyzer system Fragment Analyzer system TapeStation system Femto Pulse system
High Sensitivity DNA kit
(p/n 5067–4626)
NGS Fragment kit
(p/n DNF-473)
D1000 ScreenTape assay
(p/n 5067–5582 and
5067-5583)
Ultra Sensitivity NGS kit
(p/n FP-1101)
May also use: HS NGS Fragment kit
(p/n DNF-474)
D5000 ScreenTape assay
(p/n 5067–5588 and
5067-5589)
DNA 1000 kit
(p/n 5067–1504)
HS Small Fragment kit
(DNF-477)
High Sensitivity D1000 ScreenTape assay
(p/n 5067–5584 and
5067-5585)
DNA 7500 kit
(p/n 5067–1506)
High Sensitivity D5000 ScreenTape assay
(p/n 5067–5592 and
5067-5593)
DNA 12000 kit
(p/n 5067–1508)

For Research Use Only. Not for use in diagnostic procedures.

Long-Read Sequencing

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.
  • The TapeStation systems provide fast analysis of samples up to 60,000 bp in less than 2 minutes per sample with the Genomic DNA ScreenTape assay (p/n 5067–5365 and 5067-5366).

Femto Pulse system

Long-read sequencing library separated on the Femto Pulse system using the Genomic DNA 165 kb kit (FP-1002).

Long-read sequencing library separated on the Femto Pulse system

Long-read sequencing library separated using the HS Large Fragment 50 kb kit

Fragment Analyzer system

Long-read sequencing library separated using the HS Large Fragment 50 kb kit. Capillary electrophoresis was performed on a Fragment Analyzer equipped with a short capillary array, 33 cm.

TapeStation system

Electropherogram of reference gDNA analyzed on a TapeStation system using the DNA ScreenTape assay. With a DIN of 9.7, the sample qualifies as starting material for DNA library preparation workflows.

gDNA analyzed on a TapeStation system

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.

Instrument Part Number(s) Reagent Kit Name Sizing Range Quantification Range*
2100 Bioanalyzer system 5067–4626 High Sensitivity DNA kit 50 to 7,000 bp Fragment: 5 to 500 pg/µL
Smear: 0.1 to 10 ng/µL
5067–1504 DNA 1000 kit 25 to 1,000 bp 0.5 to 50 ng/µL
5067–1506 DNA 7500 kit 100 to 7,500 bp 0.5 to 50 ng/µL
5067–1508 DNA 12000 kit 100 to 12,000 bp 0.5 to 50 ng/µL
Fragment Analyzer systems DNF-473-0500
DNF-473-1000
NGS Fragment kit 100 to 6,000 bp Fragment: 0.1 to 10 ng/µL
Smear: 5 to 100 ng/µL
DNF-474-0500
DNF-474-1000
HS NGS Fragment kit 100 to 6,000 bp Fragment: 5 to 500 pg/µL
Smear: 50 to 5,000 pg/µL
DNF-477-0500
DNF-477-1000
HS Small Fragment kit 50 to 1,500 bp Fragment: 5 to 500 pg/µL>
Smear: 100 to 5,000 pg/µL
DNF-464-0500 HS Large Fragment 50 kb kit 75 to 50,000 bp Fragment: 5 to 600 pg/µL
Smear: 50 pg/µL to 5 ng/µL
DNF-492-0500
DNF-492-1000
Large Fragment kit 50 to 20,000 bp Fragment: 0.1 to 10 ng/µL
Smear: 5 to 100 ng/µL
TapeStation systems 5067-5582 (ScreenTape)
5067-5583 (Reagents)
D1000 ScreenTape 35 to 1,000 bp Fragment: 0.1 to 50 ng/µL
Smear: 5 to 100 ng/µL
5067-5584 (ScreenTape)
5067-5585 (Reagents)
High Sensitivity D1000 ScreenTape 35 to 1,000 bp Fragment: 10 to 1,000 pg/µL
Smear: 0.5 to 15 ng/µL
5067-5588 (ScreenTape)
5067-5589 (Reagents)
D5000 ScreenTape 100 to 5,000 bp 0.1 to 50 ng/µL
5067-5592 (ScreenTape)
5067-5593 (Reagents)
High Sensitivity D5000 ScreenTape 100 to 5,000 bp 10 to 1,000 pg/µL
5067-5565 (ScreenTape)
5067-5566 (Reagents)
Genomic DNA ScreenTape 200 to >60,000 bp 10 to 100 ng/µL
Femto Pulse system FP-1101-0275 Ultra Sensitivity NGS kit 100 to 6,000 bp Fragment: 0.05 to 5 pg/µL
Smear: 5 to 250 pg/µL
FP-1002-0275 Genomic DNA 165 kb kit 1.3 to 165 kb Fragment: 0.3 to 30 pg/µL
Smear: 5 to 500 pg/µL

For Research Use Only. Not for use in diagnostic procedures.

NGS Application Notes

Comparison of the Agilent HS Small Fragment Kit and the Agilent HS NGS Fragment Kit on the Agilent 5200 Fragment Analyzer System

The Agilent Fragment Analyzer equipped with the short or ultra-short array offers consistent sizing and quantification of gDNA smears.

Quality Control of Magnis SureSelect XT HS Workflow with Agilent Automated Electrophoresis Solutions

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.

Monitoring Library Preparation for Next-Generation Sequencing in systems Biology Omics Analysis

Evaluation of the Agilent 4150 TapeStation system with ScreenTape assay to monitor exome and transcriptome library preparation for systems biology omics analysis.

Sample Quality Control in Agilent NGS Solutions

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

Sequencing Workflows Using the TapeStation System

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.

For Research Use Only. Not for use in diagnostic procedures.