Tags

Type your tag names separated by a space and hit enter

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging.
BMC Genomics. 2014 Oct 06; 15:864.BG

Abstract

BACKGROUND

Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.

RESULTS

Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.

CONCLUSIONS

The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

Authors+Show Affiliations

No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableU, S, Military HIV Research Program (MHRP), Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, USA. rthomas@hivresearch.org.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

25283548

Citation

Ehrenberg, Philip K., et al. "High-throughput Multiplex HLA Genotyping By Next-generation Sequencing Using Multi-locus Individual Tagging." BMC Genomics, vol. 15, 2014, p. 864.
Ehrenberg PK, Geretz A, Baldwin KM, et al. High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging. BMC Genomics. 2014;15:864.
Ehrenberg, P. K., Geretz, A., Baldwin, K. M., Apps, R., Polonis, V. R., Robb, M. L., Kim, J. H., Michael, N. L., & Thomas, R. (2014). High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging. BMC Genomics, 15, 864. https://doi.org/10.1186/1471-2164-15-864
Ehrenberg PK, et al. High-throughput Multiplex HLA Genotyping By Next-generation Sequencing Using Multi-locus Individual Tagging. BMC Genomics. 2014 Oct 6;15:864. PubMed PMID: 25283548.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging. AU - Ehrenberg,Philip K, AU - Geretz,Aviva, AU - Baldwin,Karen M, AU - Apps,Richard, AU - Polonis,Victoria R, AU - Robb,Merlin L, AU - Kim,Jerome H, AU - Michael,Nelson L, AU - Thomas,Rasmi, Y1 - 2014/10/06/ PY - 2014/04/11/received PY - 2014/09/23/accepted PY - 2014/10/7/entrez PY - 2014/10/7/pubmed PY - 2015/7/3/medline SP - 864 EP - 864 JF - BMC genomics JO - BMC Genomics VL - 15 N2 - BACKGROUND: Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq. RESULTS: Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method. CONCLUSIONS: The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run. SN - 1471-2164 UR - https://www.unboundmedicine.com/medline/citation/25283548/High_throughput_multiplex_HLA_genotyping_by_next_generation_sequencing_using_multi_locus_individual_tagging_ L2 - https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-15-864 DB - PRIME DP - Unbound Medicine ER -