Ligation-based library preparation

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.
  

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).

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