Next-generation sequencing of HLA-G based on long-range polymerase chain reaction

TY - JOUR

T1 - Next-generation sequencing of HLA-G based on long-range polymerase chain reaction

AU - Nilsson, L. L.

AU - Funck, T.

AU - Kjersgaard, N. D.

AU - Hviid, T. V.F.

PY - 2018

Y1 - 2018

N2 - Clarifying the functional roles of HLA-G and the variation in the HLA-G gene that affects the expression are increasingly important in reproduction, cancer, organ transplantation, and autoimmune diseases. The homology between HLA genes and the genetic variability within each gene complicates the design of HLA gene-specific genotyping assays. We have designed a high-throughput, cost-efficient, robust, and specific assay for sequencing the full HLA-G gene including the 5′-upstream regulatory region, introns, and the 3′-untranslated region, using the next-generation sequencing (NGS) platform Ion Torrent PGM (Thermo Fisher Scientific, Waltham, Massachusetts). Conventional sequencing methods require the design of multiple primer pairs in order to cover the entire HLA-G gene. Designing multiple primer pairs specific for the HLA-G gene that also target all known alleles is difficult. Here, we present a setup that by the use of long-range polymerase chain reaction amplifies the whole HLA-G gene in a single reaction, which only requires a single HLA-G-specific primer pair. Enzymatic DNA shearing is used to break the long-range PCR product into shorter fragments ranging from 75 to 200 bp in length that are sequenced by NGS.

AB - Clarifying the functional roles of HLA-G and the variation in the HLA-G gene that affects the expression are increasingly important in reproduction, cancer, organ transplantation, and autoimmune diseases. The homology between HLA genes and the genetic variability within each gene complicates the design of HLA gene-specific genotyping assays. We have designed a high-throughput, cost-efficient, robust, and specific assay for sequencing the full HLA-G gene including the 5′-upstream regulatory region, introns, and the 3′-untranslated region, using the next-generation sequencing (NGS) platform Ion Torrent PGM (Thermo Fisher Scientific, Waltham, Massachusetts). Conventional sequencing methods require the design of multiple primer pairs in order to cover the entire HLA-G gene. Designing multiple primer pairs specific for the HLA-G gene that also target all known alleles is difficult. Here, we present a setup that by the use of long-range polymerase chain reaction amplifies the whole HLA-G gene in a single reaction, which only requires a single HLA-G-specific primer pair. Enzymatic DNA shearing is used to break the long-range PCR product into shorter fragments ranging from 75 to 200 bp in length that are sequenced by NGS.

KW - HLA

KW - HLA-G

KW - long-range PCR

KW - targeted NGS

U2 - 10.1111/tan.13342

DO - 10.1111/tan.13342

M3 - Journal article

C2 - 30014615

AN - SCOPUS:85053637877

SN - 2059-2302

VL - 92

SP - 144

EP - 153

JO - HLA

JF - HLA

IS - 3

ER -