How to prepare a DNA Library
Before DNA or RNA samples can be sequenced, they must first be fragmented, end-repaired, and ligated to sequencing adapters. There are a variety of options for library preparation, and the precise protocol you use can influence your NGS sequencing results. Ligation-based library preparation is known for its high coverage uniformity, precise strand information, and reliability. The major steps of ligation-based library preparation are pictured in Figure 1 and summarized here:
- Fragmentation and end repair—Short-read sequencing technologies like those from Illumina™ cannot readily analyze very long DNA strands, so samples are fragmented into shorter, uniform pieces to prepare them for sequencing. Typically, whole genome sequencing (WGS) works best with 350 bp fragments, while hybridization capture works best with 200 bp fragments. After fragmentation, the DNA is end-repaired or end-polished. Generally, a single adenine base is added to form an overhang by an A-tailing reaction. This A overhang allows adapters containing a single thymine overhanging base to pair with the DNA fragments.
- Adapter ligation—A ligase enzyme covalently links the adapter to the DNA fragments, making a complete library molecule. Adapters serve multiple functions: they can attach the sequences to a flow cell or ensure sequencing platform-specific compatibility. They can contain barcodes—often called indexes—to identify samples and allow multiplexing in both target enrichment and sequencing. Adapters can also contain unique molecular identifiers (UMIs) which can help increase variant identification. Read more about Adapters for next generation sequencing.
- PCR amplification (optional)—Whether you amplify your libraries or not depends on the adapter type and input sample. After PCR amplification, free oligonucleotides and small fragments must be removed. PCR cleanup can be performed using magnetic beads, or a spin column.