A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing

Linh Truong, Benedict Matern, Lloyd D'Orsogna, Patricia Martinez, Marcel G.J. Tilanus, Dianne De Santis

Research output: Contribution to journalArticle

Abstract

The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100% concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.

Original languageEnglish
JournalHla
DOIs
Publication statusPublished - 16 Oct 2019

Fingerprint

Histocompatibility Testing
Gene Amplification
MHC Class I Genes
Workflow
Genes
Alleles
Exons
HLA-C Antigens
Untranslated Regions
HLA-DRB1 Chains
Polymerase Chain Reaction
MHC Class II Genes
HLA-A Antigens
HLA-B Antigens
Terminator Codon
Hematopoietic Stem Cell Transplantation
Ions
Sensitivity and Specificity
DNA

Cite this

Truong, Linh ; Matern, Benedict ; D'Orsogna, Lloyd ; Martinez, Patricia ; Tilanus, Marcel G.J. ; De Santis, Dianne. / A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing. In: Hla. 2019.
@article{e82837a5cd854d61a44171363474e7ad,
title = "A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing",
abstract = "The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100{\%} concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.",
keywords = "HLA typing, ion torrent, next generation sequencing",
author = "Linh Truong and Benedict Matern and Lloyd D'Orsogna and Patricia Martinez and Tilanus, {Marcel G.J.} and {De Santis}, Dianne",
year = "2019",
month = "10",
day = "16",
doi = "10.1111/tan.13729",
language = "English",
journal = "Hla",
issn = "2059-2302",
publisher = "John Wiley & Sons",

}

A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing. / Truong, Linh; Matern, Benedict; D'Orsogna, Lloyd; Martinez, Patricia; Tilanus, Marcel G.J.; De Santis, Dianne.

In: Hla, 16.10.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing

AU - Truong, Linh

AU - Matern, Benedict

AU - D'Orsogna, Lloyd

AU - Martinez, Patricia

AU - Tilanus, Marcel G.J.

AU - De Santis, Dianne

PY - 2019/10/16

Y1 - 2019/10/16

N2 - The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100% concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.

AB - The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100% concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.

KW - HLA typing

KW - ion torrent

KW - next generation sequencing

UR - http://www.scopus.com/inward/record.url?scp=85074596203&partnerID=8YFLogxK

U2 - 10.1111/tan.13729

DO - 10.1111/tan.13729

M3 - Article

JO - Hla

JF - Hla

SN - 2059-2302

ER -