What is next generation sequencing (NGS)?
Next generation sequencing, or high-throughput sequencing, enables sequence profiling of everything from genomes and transcriptomes to DNA-protein interactions. The technologies used are an integral part of research and discovery in biology. The ability
to generate large amounts of sequence data in a relatively short amount of time enables a wide range of applications and accelerates advances in research and the clinic, which has revolutionized understanding of human health and treatment of disease.
How are samples prepared for NGS?
Before DNA or RNA samples can be sequenced by next generation sequencing, they must be fragmented, end-repaired, and collected into adapter-ligated libraries. Library preparation protocols can influence the results generated by your NGS experiments. The
major steps of ligation-based library preparation are 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.
- Addition 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.
They can include 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 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.
Comparison of library preparation methods
There are two common methods of DNA library preparation.
Ligation: DNA fragmentation and ligation of
adapters to the ends of the fragments in two separate steps.
Tagmentation: DNA fragmentation and adapter ligation are combined into one reaction step.
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