HLA Typing with Nanopore Sequencing

The complexity and polymorphic nature of the 4 Mb region makes it challenging to resolve using short-read sequencing technology. Nanopore (ONT) sequencing, can provide long reads that overcome these challenges, enabling the identification of variants that could represent new biomarkers, unambiguous haplotype phasing of single-nucleotide variants (SNVs), and the potential for increased accuracy of HLA typing at a higher resolution.

What is HLA typing?

The human leukocyte antigen (HLA) system is a complex of genes located on the short arm chromosome 6 in humans. These genes encode class I HLA proteins (A, B, and C) and class II HLA proteins (DR, DQ, and DP) that are found on the surface of most human cells. These proteins play a crucial role in the immune system’s ability to recognize foreign substances, such as bacteria, viruses, and even transplanted organs. HLA typing is the process of determining an individual’s HLA class I and class II gene polymorphisms. High-resolution HLA genotyping utilizing PCR and NGS allows for reliable, simple, high-quality, and high-throughput analysis of the key HLA genes.

Learn more about HLA typing on Nanopore here.

NGI offer HLA typing using two different kits

Omixon NanoTYPE™

NanoTYPE™ enables the amplification of all 11 loci (HLA-A, B, C, DQA1, DQB1, DPA1, DPB1, DRB1, and DRB345) in a single long-range multiplex PCR. For targeted amplification, the NanoTYPE MONO™ kit allows amplification of individual loci. Using ONT’s library prep kit with barcodes, the kit supports multiplexing up to 96 samples per run. 

Learn more about the kit here and here.

GenDx NGS-Pronto®

GenDx NGS-Pronto kit covers 11 HLA loci (HLA-A, B, C, DRB1, DQB1, DPB1, DRB3/4/5, DQA1, DPA1) and avoids fragmentation, producing long, intact amplicons for full phasing. Using ONT’s library prep kit with barcodes, the kit supports multiplexing up to 96 samples per run.

Learn more about the kit here.

Sample requirements

• Sample type: Genomic DNA (gDNA)
• Sample amount:
  • GenDx NGS-Pronto®: 10-30 ng, in 6 µL
  • Omixon NanoTYPE™: 200 ng in 10 μL
• Sample volume:
  • We require at least 4 μL used for sample QC. This volume is in addition to the requirement stated above.
• Sample quality:
  • High-quality gDNA, >7 kb average fragment size.
• Sample origin:
  • We recommend isolating human gDNA from whole blood, however genomic DNA from other sources is acceptable.
• Sample extraction method: we recommend using a column-based kit without Phenol.
  • The samples should be resuspended in nuclease-free water or low-EDTA buffer (TE buffer)
  • Do not collect blood in heparinized tubes.
  • Do not use lipemic or hemolyzed samples or blood samples from patients undergoing heparin therapy.
  • 260/280 absorbance ratio values of 1.8-2.0 and 260/230 absorbance ratio values of 2.0-2.2. Values outside this range indicate impurities or the presence of contaminants (alcohol, salts, detergents, formaldehyde, heparin).

How to evaluate the sample quality

Even when we perform a QC in all samples, we require our users to assess both DNA quality and concentration. To accomplish this we recommend using the following:

• DNA concentration
  • Fluorometric measurements (Qubit, Quant-it)
  • Do not use absorbance measurements (Nanodrop, spectrophotometer)
• DNA quality
  • Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation)
  • Do not use gel electrophoresis

What we do with your samples

Once you sign the agreement, you can send your sample to us. Please follow our guidelines for sample submission.

Reception control

Upon receiving your samples at NGI, we begin with a Reception Control step to ensure they meet our quality requirements. If a sample does not pass this initial check, we will contact you to discuss potential solutions. For samples that pass, you will be notified, and they will be queued for library preparation

HLA amplification

The HLA loci are amplified using multiplex PCR or singleplex PCR, depending on the chosen kit.

Library Preparation

The resulting amplicons are barcoded (when multiplexing) and adapted for Nanopore sequencing.

Library QC and Sequencing

During this phase, we assess the library yield. Libraries that pass this quality control step are queued for sequencing. If we determine that a library does not meet our QC standards, we will inform you.

Expected Results

We have consistently generated libraries that yield high-quality reads for HLA typing at up to 3-field resolution, provided that the sample requirements are met. In our tests, we have even successfully resolved typing for some loci that were previously unresolved using short-read sequencing technology.

Bioinformatics

Depending on the selected kit, HLA typing is analyzed using either OMIXON NanoTYPER™ or GenDx NGSengine® software with default settings. We will deliver a QC report along with the HLA typing results and sequencing data.

Last Updated: 21st November 2024