Next-Gen Sequencing Library Prep
NGS Library Prep

As biologists began conducting larger sequencing projects, they were confronted with the limitations of Sanger sequencing, namely low throughput. Next-generation sequencing using prepared libraries was developed as a solution for the high-throughput shortcomings of Sanger sequencing.


What is Next-Gen Sequencing (NGS)?

Talk to me about my NGS workflow.

The development of chain termination sequencing in the late 1970s by Frederick Sanger (generally known as Sanger sequencing) forever changed biology, allowing researchers to determine first the sequence of a gene, and ultimately entire genomes. Next-generation sequencing (NGS) refers to various high-throughput sequencing technologies that use a wide variety of sequencing chemistries. NGS technologies can be crudely 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 much shorter, comprised of two: single-molecule real-time sequencing and nanopore sequencing.

Regardless of NGS technology used, all require a reliable nucleic acid quality control process to help ensure accurate and reproducible results. Agilent - formerly Advanced Analytical (AATI) manufactures several instruments and various reagent kits designed to meet the evolving demands of NGS. Whether the demands originate from the NGS platform, or from the sample type, Agilent - formerly AATI has the QC solution.

Initially, it was a significant achievement to sequence a fragment of a gene, let alone a genome. Since the inception of NGS platforms, DNA sequencing has rapidly evolved. Current technologies enable the sequencing of challenging sample types, providing researchers with powerful tools to address complex questions underlying biology. NGS has transformed biological research, from elucidating evolutionary relationships and redefining the concept of species, to studying cancer and genetic diseases, from the underlying mechanics to developing treatments and cures.

All NGS platforms, regardless of type, depend upon the use of high quality NGS libraries to ensure successful sequencing. An NGS library is comprised of DNA sheared to an average uniform size that has undergone further enzymatic treatment specific to each NGS platform and specific library kit. Conventional methods used to assess the quality of NGS libraries lack the necessary accuracy and precision to produce truly reliable results. Reliable QC instruments for NGS libraries must deal with varied and challenging requirements. Numerous types of NGS libraries are available now for different sequencing platforms. While each of these libraries are unique, the analytic challenges posed center around: fragment size (large or small), library concentration, and/or limited library volume.

  • PCR-free libraries
  • ChIP-Seq
  • Single-Cell Sequencing
  • RNA-Seq
  • Long-Read Sequencing
  • Small RNA Libraries
Paths to NGS Interactive Poster

Walk down the paths of NGS using our Interactive Poster. With each click, you will see all of the intersecting paths and how steps can be used on different NGS paths.

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Short-Read Sequencing

Short-read sequencing platforms, as the name suggests, use NGS libraries with a smaller average size. Illumina is perhaps the most well-known manufacturer of short-read sequencing platforms. Short-read sequencing provides deep coverage of genomes and has been utilized in the de novo assembly of the genomes of countless organisms. Successful sequencing demands the optimal loading of flow cells, in turn requiring the accurate and precise determination of average library size and concentration for molarity calculations.

Figure 1. Short-read sequencing library at 50 pg/µL separated using the DNF-474 High Sensitivity NGS Fragment Analysis Kit. Capillary electrophoresis was performed on a Fragment Analyzer equipped with a short capillary array (33-55).

The DNF-473 Standard Sensitivity NGS Fragment Analysis Kit and the DNF-474 High Sensitivity NGS Fragment Analysis Kit for the Fragment Analyzerand the 5400 Fragment Analyzer helps ensure successful sequencing with confident quantification, qualification, and sizing of short-read NGS library smears. Similarly, the FP-1101 Ultra-Sensitivity NGS Analysis Kit for the Femto Pulse System provides researchers with unbeatable quantification and qualification. Notably, the FP-1101 Ultra-Sensitivity NGS Analysis Kit is ideal for situations where the short-read NGS library concentration is low or sample conservation is otherwise crucial to the overall success of the project.

Short-read sequencing provides a variety of sub-applications to address challenging sample types and unique questions. Diminish bias in sequencing libraries with PCR-Free libraries, identify epigenetic regions, and deepen understanding of gene expression with different types of short-read sequencing libraries.


Chromatin Immunoprecipitation sequencing 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 Fragment Analyzer alleviates this restriction, dramatically decreasing the amount of sample required for quality control analysis. Using the DNF-474 High Sensitivity NGS Fragment Analysis Kit with the Fragment Analyzer or 5400 Fragment Analyzer researchers can successfully quatify ChIP-seq libraries made with as few as 10,000 cells. Furthermore, Agilent - formerly AATI worked with Diagenode to develop a low-input ChIP-seq workflow, read the "Best Workflow Practices for ChIP-seq-Analysis with Small Samples" application note.

Figure 2. ChIP-seq library prepared from 10,000 cells separated using the DNF-474 High Sensitivity NGS Fragment Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer equipped with a short capillary array (33-55).

PCR-Free Libraries

Many NGS library construction protocols require at least one PCR amplification step to ensure 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 FP-1101 Ultra-Sensitivity NGS Analysis 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 Sequencing

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 unique quality control challenges due to the small smear size and a narrow smear range.

Figure 3. Small RNA library separated using the DNF-477 High Sensitivity Small DNA Fragment Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer equipped with a short capillary array (33-55).

The precision resolution of the Fragment Analyzer and the 5400 Fragment Analyzer ameliorate issues with the accurate analysis of small RNA NGS libraries. With the DNF-477 High Sensitivity Small DNA Fragment Analysis Kit on the Fragment Analyzer or the 5400 Fragment Analyzer researchers can accurately size, quantify, and qualify small RNA NGS libraries with ease. To learn more about the analytic capabilities of the DNF-477 High Sensitivity Small DNA Fragment Analysis Kit.

Long-Read Sequencing

Long-read sequencing libraries, such as the SMRT® libraries for Pacific Biosciences sequencing platforms, are comprised of fragments with a large average size, extending to 20 Kb – 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 DNA fragments this large poses a not insignificant challenge, particularly for legacy methods of DNA fragment analysis that either time consuming or unable to reliably size, quantify, and qualify large DNA fragments.

Several quality control analysis solutions are available for large DNA fragment analysis. Three reagent kits for the Fragment Analyzer and the 5400 Fragment Analyzer provide reliable and efficient analysis of large DNA fragments: DNF-492 Standard Sensitivity Large Fragment Analysis Kit, DNF-493 High Sensitivity Large Fragment Analysis Kit, and DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit. These kits provide reliable sizing through 20 Kb, though the newest kit - DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit - provides accurate sizing and quantification of large DNA fragments through 50 Kb in about 1 hour. Pacific Biosciences recommends the use of the Fragment Analyzer and DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit at critical QC checkpoints during the SMRTbell library construction workflow.

Figure 4. Pacific Biosciences SMRTBell library separated using the DNF-464 High Sensitvity Large Fragment 50 Kb Analysis Kit. Capillary electrophoresis was performed on a Fragment Analyzer equipped with a short capillary array (33-55).

Next-Gen Sequencing (NGS) Analysis Kits

Of the various methods for nucleic acid quality control analysis, the parallel capillary electrophoresis instruments developed by Agilent - formerly AATI provide accurate and versatile analysis of DNA and RNA samples. The Fragment Analyzer, 5400 Fragment Analyzer, and the Femto Pulse System provide variable throughput, high sensitivity detection, and a broad sizing range that exceed the base requirements for reliable nucleic acid QC in NGS workflows of any type.

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