TY - JOUR
T1 - Long-Read Nanopore Sequencing Validated for Human Leukocyte Antigen Class I Typing in Routine Diagnostics
AU - Matern, Benedict M.
AU - Olieslagers, Timo I.
AU - Groeneweg, Mathijs
AU - Duygu, Burcu
AU - Wieten, Lotte
AU - Tilanus, Marcel G. J.
AU - Voorter, Christina E. M.
N1 - Publisher Copyright:
© 2020 Association for Molecular Pathology and American Society for Investigative Pathology
PY - 2020/7
Y1 - 2020/7
N2 - Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D(2) nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.
AB - Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D(2) nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.
KW - CELL TRANSPLANTATION
KW - HLA GENES
U2 - 10.1016/j.jmoldx.2020.04.001
DO - 10.1016/j.jmoldx.2020.04.001
M3 - Article
C2 - 32302780
SN - 1525-1578
VL - 22
SP - 912
EP - 919
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 7
ER -