PMID- 24355006
OWN - NLM
STAT- MEDLINE
DCOM- 20140829
LR  - 20131220
IS  - 1399-0039 (Electronic)
IS  - 0001-2815 (Linking)
VI  - 83
IP  - 1
DP  - 2014 Jan
TI  - Next-generation sequencing can reveal in vitro-generated PCR crossover products: 
      some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.
PG  - 32-40
LID - 10.1111/tan.12269 [doi]
AB  - The high-resolution human leukocyte antigen (HLA) genotyping assay that we 
      developed using 454 sequencing and Conexio software uses generic polymerase chain 
      reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance 
      DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between 
      DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing 
      property of the 454 system, were generally observed at a read depth of 5%-10% of 
      the true alleles. They usually contained at least one mismatch with the IMGT/HLA 
      database, and consequently, were easily recognizable and did not cause a problem 
      for HLA genotyping. Sometimes, however, these artifactual sequences matched a 
      rare allele and the automatic genotype assignment was incorrect. These 
      observations raised two issues: (1) could PCR conditions be modified to reduce 
      such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA 
      database be artifacts rather than true alleles? Because PCR crossing over occurs 
      during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our 
      standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, 
      crossover products were detected. In 33% of the cases, these hybrid sequences 
      corresponded to named alleles. With amplification for only 28 cycles, these 
      artifactual sequences were not detectable. To investigate whether some rare 
      alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four 
      samples obtained from the investigators who submitted the sequences. In three 
      cases, the sequences were generated from true alleles. In one case, our 454 
      sequencing revealed an error in the previously submitted sequence.
CI  - (c) 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
FAU - Holcomb, C L
AU  - Holcomb CL
AD  - Department of Human Genetics, Roche Molecular Systems, Inc., Pleasanton, CA, USA.
FAU - Rastrou, M
AU  - Rastrou M
FAU - Williams, T C
AU  - Williams TC
FAU - Goodridge, D
AU  - Goodridge D
FAU - Lazaro, A M
AU  - Lazaro AM
FAU - Tilanus, M
AU  - Tilanus M
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)
RN  - 0 (HLA-DR Antigens)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Alleles
MH  - *Artifacts
MH  - Crossing Over, Genetic/genetics
MH  - DNA/*analysis
MH  - DNA Primers
MH  - Databases, Nucleic Acid
MH  - Diagnostic Errors/prevention & control
MH  - Exons
MH  - Genotype
MH  - HLA-DR Antigens/*genetics
MH  - High-Throughput Nucleotide Sequencing
MH  - *Histocompatibility Testing
MH  - Humans
MH  - Polymerase Chain Reaction/*methods/trends
MH  - Sequence Analysis, DNA
OTO - NOTNLM
OT  - IMGT HLA database
OT  - PCR crossover
OT  - human leukocyte antigen
OT  - next-generation sequencing
EDAT- 2013/12/21 06:00
MHDA- 2014/08/30 06:00
CRDT- 2013/12/21 06:00
PHST- 2013/07/03 00:00 [received]
PHST- 2013/10/10 00:00 [revised]
PHST- 2013/11/15 00:00 [accepted]
PHST- 2013/12/21 06:00 [entrez]
PHST- 2013/12/21 06:00 [pubmed]
PHST- 2014/08/30 06:00 [medline]
AID - 10.1111/tan.12269 [doi]
PST - ppublish
SO  - Tissue Antigens. 2014 Jan;83(1):32-40. doi: 10.1111/tan.12269.