PMID- 23398507
OWN - NLM
STAT- MEDLINE
DCOM- 20130917
LR  - 20130212
IS  - 1399-0039 (Electronic)
IS  - 0001-2815 (Linking)
VI  - 81
IP  - 3
DP  - 2013 Mar
TI  - High throughput HLA genotyping using 454 sequencing and the Fluidigm Access 
      Array System for simplified amplicon library preparation.
PG  - 141-9
LID - 10.1111/tan.12071 [doi]
AB  - The human leukocyte antigen (HLA) class I and class II loci are the most 
      polymorphic genes in the human genome; distinguishing the thousands of HLA 
      alleles is challenging. Next generation sequencing of exonic amplicons with the 
      454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides 
      high resolution, high throughput HLA genotyping for eight class I and class II 
      loci. HLA typing of potential donors for unrelated bone marrow donor registries 
      typically uses a subset of these loci at high sample throughput and low cost per 
      sample. The Fluidigm Access Array System enables the incorporation of 48 
      different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples 
      with up to 48 different primer pairs in a microfluidic device generating 2304 
      parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. 
      During genomic PCR, in this 4-primer system, the outer set of primers containing 
      the MID and the 454 adaptor sequences are incorporated into an amplicon generated 
      by the inner HLA target-specific primers each containing a common sequence tag at 
      the 5' end of the forward and reverse primers. Pools of the resulting amplicons 
      are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX 
      System, followed by genotyping with Conexio software. We have genotyped 192 
      samples with 100% concordance to known genotypes using 8 primer pairs (covering 
      exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs 
      in a single GS FLX run. An average of 166 reads per amplicon was obtained. We 
      have also genotyped 96 samples at high resolution (14 primer pairs covering exons 
      2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and 
      exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.
CI  - (c) 2013 John Wiley & Sons A/S.
FAU - Moonsamy, P V
AU  - Moonsamy PV
AD  - Roche Molecular Systems, Inc., Pleasanton, CA, USA. Priscilla.Moonsamy@roche.com
FAU - Williams, T
AU  - Williams T
FAU - Bonella, P
AU  - Bonella P
FAU - Holcomb, C L
AU  - Holcomb CL
FAU - Hoglund, B N
AU  - Hoglund BN
FAU - Hillman, G
AU  - Hillman G
FAU - Goodridge, D
AU  - Goodridge D
FAU - Turenchalk, G S
AU  - Turenchalk GS
FAU - Blake, L A
AU  - Blake LA
FAU - Daigle, D A
AU  - Daigle DA
FAU - Simen, B B
AU  - Simen BB
FAU - Hamilton, A
AU  - Hamilton A
FAU - May, A P
AU  - May AP
FAU - Erlich, H A
AU  - Erlich HA
LA  - eng
PT  - Journal Article
PL  - England
TA  - Tissue Antigens
JT  - Tissue antigens
JID - 0331072
RN  - 0 (DNA Primers)
SB  - IM
MH  - Cell Line
MH  - DNA Primers/metabolism
MH  - *Gene Library
MH  - Genotyping Techniques/*methods
MH  - High-Throughput Nucleotide Sequencing/*methods
MH  - Histocompatibility Testing/*methods
MH  - Humans
MH  - Microfluidics/*methods
MH  - Polymerase Chain Reaction
MH  - Sequence Analysis, DNA/*methods
MH  - Software
EDAT- 2013/02/13 06:00
MHDA- 2013/09/18 06:00
CRDT- 2013/02/13 06:00
PHST- 2012/10/29 00:00 [received]
PHST- 2013/01/17 00:00 [revised]
PHST- 2013/01/21 00:00 [accepted]
PHST- 2013/02/13 06:00 [entrez]
PHST- 2013/02/13 06:00 [pubmed]
PHST- 2013/09/18 06:00 [medline]
AID - 10.1111/tan.12071 [doi]
PST - ppublish
SO  - Tissue Antigens. 2013 Mar;81(3):141-9. doi: 10.1111/tan.12071.