Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.
BACKGROUND
A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.


RESULTS
We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.


CONCLUSIONS
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.
Vinzenz
V
Lange
DKMS Life Science Lab, Fiedlerstrasse 34, 01307 Dresden, Germany. boehme@dkms-lab.de.
Irina
I
Böhme
Jan
J
Hofmann
Kathrin
K
Lang
Jürgen
J
Sauter
Bianca
B
Schöne
Patrick
P
Paul
Viviane
V
Albrecht
Johanna M
JM
Andreas
Daniel M
DM
Baier
Jochen
J
Nething
Ulf
U
Ehninger
Carmen
C
Schwarzelt
Julia
J
Pingel
Gerhard
G
Ehninger
Alexander H
AH
Schmidt
D000483:Alleles; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D017931:DNA Primers / Q000378:metabolism; D005091:Exons; D006680:HLA Antigens / Q000235:genetics*; D006650:Histocompatibility Testing / Q000191:economics / Q000295:instrumentation*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D006680:HLA Antigens; D004247:DNA