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Updated: Mar 8, 2026
Detection of Copy Number Alterations Using Single Cell Sequencing
Published on: February 17, 2017
Typing and copy number determination for HLA-DRB3, -DRB4 and -DRB5 from next-generation sequencing data
Y Zhang1,2,3, Y Song2,3, H Cao2,3
1State Key Lab of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
Insights
A new method accurately types Human Leukocyte Antigen (HLA)-DRB3/4/5 genes using next-generation sequencing (NGS) data. This approach enhances HLA typing for disease and population studies.
Area of Science:
- Immunogenetics
- Genomics
- Bioinformatics
Background:
- Human Leukocyte Antigen (HLA)-DRB3, DRB4, and DRB5 (DRB3/4/5) are paralogs of HLA-DRB1 with critical roles in transplantation and disease association.
- Current next-generation sequencing (NGS) based HLA typing methods for DRB3/4/5 exhibit limitations, including reliance on polymerase chain reaction (PCR) and suboptimal accuracy.
Purpose of the Study:
- To develop and validate a novel, accurate HLA typing method for DRB3/4/5 using NGS data.
- To determine the copy number of DRB3/4/5 genes.
- To investigate the population frequency of DRB3/4/5 alleles.
Main Methods:
- A novel HLA typing method for DRB3/4/5 was developed, utilizing read mapping and haplotype assembly from NGS data.
- Copy number determination for DRB3/4/5 was achieved using k-means clustering based on sequencing depth ratios relative to DRB1.
- The method was validated on simulated data and real-world MHC capture Illumina sequencing data.
Main Results:
- High accuracy was achieved: 100% on simulated data and 95.88%–99.34% on real sequencing data at 4-digit resolution.
- Analysis of 1000 Genomes Project data revealed significantly lower DRB4 frequencies in African populations compared to other continental groups.
- The method successfully determined DRB3/4/5 profiles across diverse populations.
Conclusions:
- The developed method offers high accuracy for DRB3/4/5 typing, serving as a valuable complement to existing HLA typing techniques.
- This method has significant potential for applications in disease association studies, clinical transplantation, and understanding human population genetics.
- The findings contribute to a better understanding of HLA diversity and its implications in various biological and medical fields.
Background:
HLA-DRB3, DRB4 and DRB5 (DRB3/4/5) are paralogues of HLA-DRB1. They have important roles in transplantation and have been reported to be related to many diseases. HLA typing methods for DRB3/4/5 based on NGS data have many limitations now, such as need of polymerase chain reaction (PCR) or low accuracy.
Materials And Methods:
We present a HLA typing method for DRB3/4/5 based on read mapping and haplotype assembly from NGS data. Also, copy number of DRB3/4/5 is determined by a k-means clustering method according to ratio of sequencing depth between DRB3/4/5 and DRB1.
Results:
We achieved 100%, 100%, 100% accuracy on simulated data and 95.88%, 98.89%, 99.34% accuracy on MHC capture Illumina sequencing data at 4-digit resolution with 30-fold coverage for DRB3/4/5 separately. We also explored the DRB3/4/5 profiles in five continental populations through low coverage WGS data generated by the 1000 Genome Project. We found that frequency of DRB4 in African were significantly lower than that in all other populations.
Conclusion:
Our method for DRB3/4/5 typing has high accuracy. It is a good supplement to regular HLA typing and could help in disease studies, medical applications and human population diversity studies.
