# Non-invasive comparative study of HLA genotyping between urinary and blood DNA using sequencing-based typing and third-generation sequencing

## TL;DR

This study shows that DNA from urine can be used as accurately as blood DNA for HLA genotyping, offering a non-invasive alternative.

## Contribution

The study demonstrates that urinary DNA can achieve 100% concordance with blood DNA for HLA genotyping using optimized protocols.

## Key findings

- Urinary DNA showed 100% concordance with blood DNA for HLA genotyping across five loci.
- Third-generation sequencing resolved full-length HLA sequences from urinary DNA at ≥30× coverage.
- Optimized protocols enabled dual-platform validation, making urine a viable alternative to blood for HLA profiling.

## Abstract

To evaluate the feasibility of urinary DNA as a noninvasive alternative for high-resolution HLA genotyping and validate its concordance with conventional blood-based methods.

Matched urine and blood samples were collected from 11 healthy volunteers. Urinary DNA was extracted using an optimized column-based protocol, while blood DNA was processed via an automated system. High-resolution HLA typing for HLA-A, -B, -C, -DRB1, and -DQB1 loci was performed using sequencing-based typing (SBT) and third-generation sequencing (TGS), with concordance rates assessed between sample types.

The average concentration of urinary DNA exhibited significantly lower concentrations than blood DNA (9.74 ± 10.52 vs. 33.13 ± 26.78 ng/μL, p = 0.001) but comparable purity (OD 260/280 ratio: 1.65 ± 0.4 vs. 1.81 ± 0.13, p = 0.068). Remarkably, both SBT and TGS achieved 100 % concordance between urine- and blood-derived genotypes across five classical HLA loci, with TGS resolving full-length HLA sequences (5′UTR–3′UTR) at ≥30× coverage.

Urinary DNA achieves blood-comparable accuracy in noninvasive HLA genotyping. Our optimized protocol overcomes DNA yield and purity limitations through dual-platform validation (SBT/TGS), establishing urine as a clinically viable alternative for HLA profiling, especially where blood sampling is unfeasible.