Human leukocyte antigen (HLA) plays a significant role in the ability of the immune system to recognize and remove invasive, foreign, infected, and malfunctioning cells to fight disease and maintain overall health. HLA mutations can produce an aberrant immune response and have been associated with autoimmune disorders, cancer, transplant rejection, and drug sensitivity. Sequencing the HLA region can provide critical insight into various immune disorders. Unfortunately, HLA sequencing has been difficult due to the high levels of sequence homology and dense variability found within this region of the genome.
Technologies developed over the last several years, cumulatively called Next Generation Sequencing (NGS), have provided new opportunities that allow the complete characterization of the HLA genes in haploid fashion. NGS has two distinct features, 1) clonal sequencing of DNA fragments, and 2) tremendously high throughput. NGS provides the capability to phase polymorphisms thereby eliminating all ambiguities and provides HLA typing at the three to four field level without reflexive testing, thereby introducing a potentially total solution to the HLA typing problem. Quick Biology’s HLA typing takes advantage of this technology and combines long-range PCR amplification of HLA genes with sequencing on the Illumina MiSeq platform. More specifically the HLA genes A, B, C, DPA1, DQA1, and DQB1 are amplified for their entire coding length, including elements of the 5’ and 3’ untranslated regions, while DRB1, DRB3, and DRB4 are amplified from intron 1 to intron 4, and DRB5 and DPB1 are amplified from intron 1 to the 3’ untranslated region. Therefore, NGS-based HLA-typing provides high-resolution, unambiguous, and phase-resolved results with quick turnaround time.
Quick Biology is now offering two types of NGS-based HLA typing solutions and an NGS-based ABO typing:
(1) Illumina TruSight® HLA v2 Sequencing Panel
- Provides unambiguous, phase-resolved HLA typing (Table 1) in a single assay using proven Illumina NGS technology.
- Illumina TruSight® HLA v2: An Integrated DNA-to-Report Workflow for HLA Typing—The TruSight HLA v2 Sequencing Panel enables accurate and efficient HLA typing going from sample to report with fast turnaround time.
- High-Accuracy HLA Typing: TruSight HLA v2 typing results show high accuracy (A) and low ambiguity (B) compared to available reference typing results.
(2) Omixon Holotype HLA typing assay
- Leverages the power of Next Generation Sequencing (NGS) for typing of up to 11 loci (with the option of 5, 7, or 11 loci), and is licensed from The Children’s Hospital of Philadelphia (CHOP). It is also combined with HLA TwinTM software for automated analysis of ultra-high resolution with almost zero ambiguity.
- An Integrated DNA-to-Report Workflow for HLA Typing—The Omixon Holotype HLA also enables economical, accurate and efficient HLA typing going from sample to report with fast turnaround time.
- High-Accuracy HLA Typing: Omixon Holotype HLA typing results also show high accuracy and low ambiguity (* Cause by a known limitation of DRB4; ** Caused by phasing ambiguity due to a long homozyguous intronic region in DPB1 and by the lack of exon 1 coverage in DRB1).
(3) NGS-BASED ABO Typing
- A long-range amplification method covering Exons 2-7 of the ABO gene, and the resulting libraries can be sequenced on Illumina instruments with 2x150bp paired-end reads.
- The ABO assay leverages the power of the Holotype HLA™ workflow, providing high throughput and allele level resolution genotyping of the ABO gene.
· HLA Typing needs > 0.8 ug of gDNA or samples from whole blood, blood cells (B-cell lines, buffy coats, cord blood or any fraction of white blood cells), saliva and buccal swabs. ABO Typing needs > 100 ng of gDNA.
· gDNA should be dissolved in water as the EDTA in TE buffer can inhibit long-range PCR reactions and its recommended concentration is 20 - 30ng/μl. We highly recommend using a fluorescence-based quantitation method to determine the gDNA concentration.
· A 260nm/280nm absorbance ratio between 1.7 and 1.9. A 260nm/230nm absorbance ratio of 1.7 or greater.
· Minimal degradation. DNA that is old or has gone through repeated freeze/thaws will suffer from more degradation.
About 2-4 weeks, depends on the quality of submitted samples.