Next generation sequencing (NGS) is a new methodology for high frequency throughput for HLA alleles with the highest resolution possible. NGS is currently only performed in a few laboratories in the United States for HLA. NGS amplifies the entire region of the MHC (UTRs, introns, and exons) as opposed to SSO and SSP which are exon sequences.
NGS setup is complicated and may take many days to generate results.
To start, DNA is amplified by PCR using a cocktail of dNTPs, primers, and polymerase. After PCR, the amplicons must be fragmented, the fragmented ends must be repaired, and then ligated to a special adaptor for testing onto a flow cell. A unique feature of NGS involves forming a "bridge of amplified DNA" on the flow cell that forms detectable clusters via a camera as each cycle of nucleotide is added. Each nucleotide is labeled with a specific color. Terminators are also present, which, if they attach, stop elongation so that the exact sequence can be determined by software. As one nucleotide is added, a camera takes a picture. If a nucleotide is added, it fluoresces a certain color based on the nucleotide added, which is similar to Sanger's sequencing but more robust.
NGS is great for resolving low resolution ambiguities and discovering new alleles because the sequences can be compared to known databases that contain currently characterized human HLA sequences.
