PMID- 25283548
OWN - NLM
STAT- MEDLINE
DCOM- 20150702
LR  - 20211021
IS  - 1471-2164 (Electronic)
IS  - 1471-2164 (Linking)
VI  - 15
IP  - 1
DP  - 2014 Oct 6
TI  - High-throughput multiplex HLA genotyping by next-generation sequencing using 
      multi-locus individual tagging.
PG  - 864
LID - 10.1186/1471-2164-15-864 [doi]
LID - 864
AB  - 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.
FAU - Ehrenberg, Philip K
AU  - Ehrenberg PK
FAU - Geretz, Aviva
AU  - Geretz A
FAU - Baldwin, Karen M
AU  - Baldwin KM
FAU - Apps, Richard
AU  - Apps R
FAU - Polonis, Victoria R
AU  - Polonis VR
FAU - Robb, Merlin L
AU  - Robb ML
FAU - Kim, Jerome H
AU  - Kim JH
FAU - Michael, Nelson L
AU  - Michael NL
FAU - Thomas, Rasmi
AU  - Thomas R
AD  - U, S, Military HIV Research Program (MHRP), Walter Reed Army Institute of 
      Research, 503 Robert Grant Avenue, Silver Spring, MD, USA. 
      rthomas@hivresearch.org.
LA  - eng
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20141006
PL  - England
TA  - BMC Genomics
JT  - BMC genomics
JID - 100965258
RN  - 0 (HLA-A Antigens)
RN  - 0 (HLA-B Antigens)
RN  - 0 (HLA-C Antigens)
RN  - 0 (HLA-DRB1 Chains)
SB  - IM
MH  - Genotyping Techniques/methods
MH  - HLA-A Antigens/*genetics
MH  - HLA-B Antigens/*genetics
MH  - HLA-C Antigens/*genetics
MH  - HLA-DRB1 Chains/*genetics
MH  - High-Throughput Nucleotide Sequencing
MH  - Histocompatibility Testing/*methods/trends
MH  - Humans
MH  - Sensitivity and Specificity
MH  - Sequence Analysis, DNA
PMC - PMC4196003
EDAT- 2014/10/07 06:00
MHDA- 2015/07/03 06:00
PMCR- 2014/10/06
CRDT- 2014/10/07 06:00
PHST- 2014/04/11 00:00 [received]
PHST- 2014/09/23 00:00 [accepted]
PHST- 2014/10/07 06:00 [entrez]
PHST- 2014/10/07 06:00 [pubmed]
PHST- 2015/07/03 06:00 [medline]
PHST- 2014/10/06 00:00 [pmc-release]
AID - 1471-2164-15-864 [pii]
AID - 6530 [pii]
AID - 10.1186/1471-2164-15-864 [doi]
PST - epublish
SO  - BMC Genomics. 2014 Oct 6;15(1):864. doi: 10.1186/1471-2164-15-864.