PMID- 32324777
OWN - NLM
STAT- MEDLINE
DCOM- 20200720
LR  - 20240425
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 15
IP  - 4
DP  - 2020
TI  - Performance of a multiplexed amplicon-based next-generation sequencing assay for 
      HLA typing.
PG  - e0232050
LID - 10.1371/journal.pone.0232050 [doi]
LID - e0232050
AB  - BACKGROUND: Next-generation sequencing (NGS) has enabled efficient 
      high-resolution typing of human leukocyte antigen (HLA) genes with minimal 
      ambiguity. Most commercially available assays amplify individual or subgroup of 
      HLA genes by long-range PCR followed by library preparation and sequencing. The 
      AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA 
      genes in one PCR reaction. Here, we report the performance of this unique 
      workflow evaluated using 218 genetically diverse samples. METHODS: Five whole 
      genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/DRB345/DQB1/DPB1; 
      excluding exon 1 and part of intron 1) were amplified in a multiplexed, 
      long-range PCR. Manual library preparation was performed per manufacturer's 
      protocol, followed by template preparation and chip loading on the Ion Chef, and 
      sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and 
      repeat testing were followed; technologists were blinded to the reference 
      results. The concordance between AllType and reference results was determined at 
      2-field resolution. We also describe the ranges of input DNA and library 
      concentrations, read number per sample and per locus, and key health metrics in 
      relation to typing results. RESULTS: The concordance rates were 98.6%, 99.8% and 
      99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, 
      respectively. Three genotypes were discordant, all of which shared the same G 
      group typing results with the reference. Most ambiguous genotypes (116 out of 
      144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for 
      HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and 
      library concentrations were tolerated. Per sample read numbers were adequate for 
      accurate genotyping. Per locus read numbers showed some inter-lot variations, and 
      a trend toward improved inter-locus balance was observed with later lots of 
      reagents. CONCLUSION: The AllType assay on the Ion Chef/Ion S5 platform offers a 
      robust and efficient workflow for clinical HLA typing at the 2-field resolution. 
      The multiplex PCR strategy simplifies the laboratory procedure without 
      compromising the typing accuracy.
FAU - Liu, Chang
AU  - Liu C
AUID- ORCID: 0000-0001-6624-1454
AD  - Division of Laboratory and Genomic Medicine, Department of Pathology and 
      Immunology, School of Medicine, Washington University in St. Louis, St. Louis, 
      Missouri, United States of America.
FAU - Duffy, Brian F
AU  - Duffy BF
AD  - HLA Laboratory, Barnes-Jewish Hospital, St. Louis, Missouri, United States of 
      America.
FAU - Weimer, Eric T
AU  - Weimer ET
AUID- ORCID: 0000-0003-3677-5724
AD  - Department of Pathology & Laboratory Medicine, University of North Carolina at 
      Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States of 
      America.
AD  - Molecular Immunology Laboratory, McLendon Clinical Laboratories, UNC Hospitals, 
      Chapel Hill, North Carolina, United States of America.
FAU - Montgomery, Maureen C
AU  - Montgomery MC
AD  - Molecular Immunology Laboratory, McLendon Clinical Laboratories, UNC Hospitals, 
      Chapel Hill, North Carolina, United States of America.
FAU - Jennemann, Jo-Ellen
AU  - Jennemann JE
AD  - HLA Laboratory, Barnes-Jewish Hospital, St. Louis, Missouri, United States of 
      America.
FAU - Hill, Rachel
AU  - Hill R
AD  - HLA Laboratory, Barnes-Jewish Hospital, St. Louis, Missouri, United States of 
      America.
FAU - Phelan, Donna
AU  - Phelan D
AD  - HLA Laboratory, Barnes-Jewish Hospital, St. Louis, Missouri, United States of 
      America.
FAU - Lay, Lindsay
AU  - Lay L
AD  - HLA Laboratory, Barnes-Jewish Hospital, St. Louis, Missouri, United States of 
      America.
FAU - Parikh, Bijal A
AU  - Parikh BA
AUID- ORCID: 0000-0003-2490-8294
AD  - Division of Laboratory and Genomic Medicine, Department of Pathology and 
      Immunology, School of Medicine, Washington University in St. Louis, St. Louis, 
      Missouri, United States of America.
LA  - eng
GR  - R41 AI142919/AI/NIAID NIH HHS/United States
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20200423
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (HLA Antigens)
SB  - IM
MH  - HLA Antigens/*genetics
MH  - High-Throughput Nucleotide Sequencing/*methods
MH  - Histocompatibility Testing/*methods
MH  - Humans
MH  - Multilocus Sequence Typing
MH  - Multiplex Polymerase Chain Reaction/methods
MH  - Reproducibility of Results
MH  - Sequence Analysis, DNA/methods
MH  - Tissue Donors
MH  - Workflow
PMC - PMC7179861
COIS- The authors have declared that no competing interests exist.
EDAT- 2020/04/24 06:00
MHDA- 2020/07/21 06:00
PMCR- 2020/04/23
CRDT- 2020/04/24 06:00
PHST- 2019/12/16 00:00 [received]
PHST- 2020/04/06 00:00 [accepted]
PHST- 2020/04/24 06:00 [entrez]
PHST- 2020/04/24 06:00 [pubmed]
PHST- 2020/07/21 06:00 [medline]
PHST- 2020/04/23 00:00 [pmc-release]
AID - PONE-D-19-34784 [pii]
AID - 10.1371/journal.pone.0232050 [doi]
PST - epublish
SO  - PLoS One. 2020 Apr 23;15(4):e0232050. doi: 10.1371/journal.pone.0232050. 
      eCollection 2020.