Illumina vs Nanopore Sequencing: A Technical Comparison for HLA Typing

Introduction

Human leukocyte antigen (HLA) typing is essential for bone marrow and organ transplantation. Accurate HLA typing at high resolution is critical to ensure donor-recipient matching and prevent graft rejection. Two major sequencing technologies used for HLA typing are Illumina short-read sequencing and Oxford Nanopore long-read sequencing. This whitepaper provides a technical overview of these platforms and compares their utility for HLA typing.

Overview of Sequencing Platforms

Illumina Short-Read Sequencing

Illumina sequencing utilizes sequencing-by-synthesis where labeled nucleotides are detected as they are incorporated into growing DNA strands. This produces short reads, typically 100-250 basepairs in length. Strengths of Illumina include very high accuracy (Q30 scores >80%), high throughput, and low cost. However, short reads make it challenging to resolve complex genomic regions.

Oxford Nanopore Long-Read Sequencing

Oxford Nanopore uses nanopores and electrical current changes to sequence long strands of DNA. Read lengths range from 1 kb to over 2 mb. Benefits include ultralong reads to span repetitive regions, simple library prep, and real-time data output. Drawbacks are higher error rates of 5-15% and lower throughput versus Illumina.

HLA Typing Comparison

Both Illumina and Nanopore can accurately type HLA genes which are highly polymorphic. However, key differences impact typing resolution.

Read Length

The long reads from Nanopore sequencing can span entire HLA genes and haplotypes. This simplifies phasing of polymorphisms across each gene. In contrast, short Illumina reads must be computationally phased which is complicated by the repetitive nature and high polymorphism of HLA.

Error Rates

The ultrahigh accuracy of Illumina provides near-perfect base resolution of HLA alleles. Nanopore historically had 5-15X higher error rates which could potentially confuse closely related alleles. In recent years, they have improved significantly on their error rates and are now comparable to the Illumina error rates. Consensus and hybrid sequencing approaches can mitigate error rates.

Cost and Time

Illumina has a higher upfront cost for instruments but very low cost per sample. Nanopore requires only a small, portable MinION device, allowing rapid in-field HLA typing. Computational analysis is faster for Illumina while Nanopore offers real-time data output. The cost per sample for HLA Typing for the Nanopore sequencing is the same or relatively close to Illumina.

Conclusions

In summary, both Illumina and Nanopore platforms have unique advantages for HLA typing. Long Nanopore reads simplify phasing across entire genes, while short, accurate Illumina reads provide precision at the basepair level. Hybrid approaches combining both methods represent a promising future direction to leverage these complementary strengths.

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