Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-12

AUTHORS

Vinzenz Lange, Irina Böhme, Jan Hofmann, Kathrin Lang, Jürgen Sauter, Bianca Schöne, Patrick Paul, Viviane Albrecht, Johanna M Andreas, Daniel M Baier, Jochen Nething, Ulf Ehninger, Carmen Schwarzelt, Julia Pingel, Gerhard Ehninger, Alexander H Schmidt

ABSTRACT

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. More... »

PAGES

63

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2164-15-63

DOI

http://dx.doi.org/10.1186/1471-2164-15-63

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1023510927

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24460756


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38 schema:description 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.
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