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Ligation-based library preparation

Overview

To prepare samples for next generation sequencing (NGS), they must be transformed and collected into libraries. The main types of library prep are ligation and tagmentation. Ligation-based library prep can be enzymatic or physical. Preparing quality libraries opens the door to discovery through a variety of NGS-based applications.

How to prepare a DNA library

Before DNA or RNA samples can be sequenced, they must be fragmented, end-repaired, and ligated to sequencing adapters. Library preparation protocols can influence the results generated by your next generation sequencing data. The major steps of ligation-based library preparation are pictured below (Figure 1) and summarized as follows:

  • Fragmentation and end repair: Short-read sequencing technologies like those from Illumina cannot readily analyze very long DNA strands, so samples are fragmented into uniform pieces to make them amenable to sequencing. After fragmentation, the DNA fragments are end repaired or end polished. Generally, a single adenine base is added to form an overhang via an A-tailing reaction. This A overhang allows adapters containing a single thymine overhanging base to base pair with the DNA fragments.
  • Ligation of adapters: A ligase enzyme covalently links the adapter and insert DNA fragments, making a complete library molecule. These adapters serve multiple functions. They attach the sequences to the flow cell to allow sequencing, and they can contain barcodes, also called indexes, to identify samples and permit multiplexing. Read more about Adapters for next generation sequencing.
  • PCR amplification (optional): Whether or not you amplify your libraries depends on the adapter type and sample input used. After PCR amplification, remaining oligonucleotides and small fragments must be removed. PCR clean-up can be performed using magnetic beads or a spin column.
Figure 1. Overview of library preparation by fragmentation. The workflow shows the steps to take you from sample to sequencing.

Why is fragmenting important?

Fragmenting shears the samples into pieces of DNA of a desired length. Typically, whole genome sequencing works best with 350 bp fragments, and hybridization capture works best with 200 bp fragments.

Methods of fragmentation

There are 2 main methods of fragmentation for ligation-based DNA library prep:

  • Physical: DNA can be sheared physically using acoustics, nebulization, centrifugal force, needles, or hydrodynamics. While these methods achieve high sensitivity and unbiased results, they require specialized machinery. For genomic DNA, or DNA derived from formalin-fixed, paraffin-embedded (FFPE) samples, we recommend using Covaris shearing to achieve average insert sizes of 150–300 base pairs. Cell-free DNA (cfDNA) typically has an average size of 160 base pairs, so no further fragmentation is required. For physical fragmentation or for samples that do not require fragmentation, we recommend the xGen Prism DNA Library Prep Kit.
  • Enzymatic: Enzymes are used to digest DNA into fragments before end repair and A-tailing. This method can sometimes introduce a GC bias that does not occur by physical fragmentation. However, enzymatic fragmentation is quick, easy, and does not require any special equipment. This method is offered by IDT in our Lotus DNA Library Prep Kit. Due to optimization of this reaction, the Lotus DNA Library Prep Kit features consistent coverage and does not show a GC bias (Figure 2).
Figure 2. Uniform genome coverage for PCR-free and PCR-amplified libraries. Libraries were prepared using 100 ng of human gDNA (NA12878, Coriell) using full-length UDI-UMI adapters (IDT) or amplified using 4 cycles of PCR and sequenced on the MiSeq® (PCR-free) and HiSeq® 4000 (amplified) platforms (Illumina). Normalized coverage of each library is shown as dark and light blue lines, the expected normalized coverage of 1.0 is indicated with a dotted line, and the number of 100 bp regions at each GC% is shown as a histogram in grey.

Alternative methods

Tagmentation is an alternative protocol that combines the fragmentation and adapter ligation steps. Some versions of this protocol could introduce a coverage bias that, depending on experimental design, may impact sequencing results.

Downstream applications

  • Whole genome sequencing (WGS)
  • PCR-free or PCR-amplified sequencing
  • Detection of germline inherited single nucleotide polymorphisms (SNPs), copy number variants (CNVs), and insertions/deletions (indels)
  • Hybridization capture of target sequences (e.g., the exome or transcripts of interest)
  • Low-frequency somatic variation detection of single nucleotide variants (SNVs), CNVs, and indels
  • RNA-seq
  • Metagenomic sequencing

Get started with library preparation solutions

Working in one of these applications? Just starting? See how you can easily improve your workflows and results.

Lotus DNA Library Prep Kit

The Lotus DNA Library Prep Kit enables streamlined preparation of high-quality next generation sequencing (NGS) libraries from double-stranded DNA (dsDNA)—generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing applications on Illumina platforms.

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xGen Prism DNA Library Prep Kit

The xGen Prism DNA Library Prep Kit empowers you with sensitive and accurate variant detection from degraded samples, such as cell-free DNA (cfDNA) or formalin-fixed, paraffin-embedded (FFPE) samples. The kit’s proprietary ligation strategy maximizes conversion and virtually eliminates adapter-dimer formation. The unique molecular identifier (UMI) sequences incorporated during single-stranded ligation enable a variety of deduplication and error correction strategies.

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