Extension of HLA allele sequences by full-length HLA allele-specific hemizygous Sanger sequencing (SSBT)
The Human Leucocyte Antigen (HLA) is one of the most polymorphic gene systems present in the human genome and due to this high polymorphism, the golden standard for typing at the allele level has been and still is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, therefore the majority of HLA alleles lack full length sequence information.
The goal of this project is to extend the sequences of as many incompletely covered HLA alleles as possible by full length unambiguous Sanger Sequencing. Although NGS approaches are currently implemented in several laboratories, many laboratories combine NGS with Sanger sequencing or use Sanger sequencing only as high resolution typing approach. For those labs that are not able to perform NGS, this project offers the possibility to actively participate in the workshop with Sanger Sequencing. Within this project full length Sanger sequencing of alleles with partially known allele sequences will be performed. The Maastricht SSBT approach enables full-length sequencing and separates the alleles based on available low-resolution typing data. The previous problem with Sanger sequencing were three kinds of ambiguities: (1) genotype ambiguity due to cis-trans polymorphism, (2) allele ambiguity due to polymorphism outside the peptide-binding groove and (3) ambiguities due to incomplete sequences. With the Maastricht approach of Sanger sequencing ambiguities (1) and (2) are resolved by hemizygous and full length sequencing, respectively, whereas the ambiguity (3) will be taken care off by the workshop to complete the sequences of as many HLA alleles as possible.
The Maastricht HLA SSBT approach is previously described (Voorter et al 2014). Based on the low resolution data the SSBT groups for group-specific full length amplification and sequencing are selected, resulting in hemizygous unambiguous allele sequences so that cis-trans phasing is not an issue. Furthermore, since this approach reveals full-length gene sequences this methodology can be used to identify and confirm new allele sequence polymorphism of alleles with unknown intron and exons. Participants will receive SSBT reagents and a detailed protocol, which after QA can be used to identify the full-length sequence of those alleles of which the sequence is not yet completely covered. All cells encountered in your laboratory from which it is known that the sequence info is incomplete (IMGT/HLA) are candidates to study. A list of these alleles can be found at the IMGT/HLA database. If you have such candidates and you are not able to perform the full length sequencing yourself, you can still participate by sending these candidates to the project leaders for inclusion in the workshop.
Voorter CE, Palusci F ,Tilanus MG. Sequence-based typing of HLA: an improved group specific full-length gene sequencing approach 2014: 101-14. In: Beksac M, ed. Methods Mol. Biol. Humana Press, New York
Goal: To extend the sequences of as many incompletely covered HLA alleles as possible by full length unambiguous Sanger Sequencing.
Participation: Investigators can participate in this project by:
- HLA SSBT typing of locally collected specimens. In 2015 the Maastricht SSBT kit for research use only (RUO) will be available to the participants. Each participant lab will test a set of five blinded DNA samples as quality control to evaluate the performance. These samples will be provided by the organization and will have to be full length sequenced. Both quality control samples and SSBT kit reagents will be provided by the IHIWS organizing committee and Maastricht group for the IHIWS SSBT project respectively, but participants will be charged for shipment fee. The laboratories performing testing are submitting raw sequencing data and genotype results electronically.
- Submission of DNA specimens including one or more incompletely covered HLA allele(s). For submission it is necessary that the specimen has previously been HLA typed. These typing data must be provided to the organization. The Maastricht Laboratory will resolve full length sequences of as many of these specimen as possible.
All full-length sequences will be submitted to the EMBL and IMGT/HLA database. The participating laboratory is the reference submitter for the nomenclature report. Details on submission will follow. In case a sample has only been full length sequence typed for a single locus by the participant, the participant needs to provide additional DNA to the IHIWS workshop to enable NGS for typing all HLA loci full length.
Project leaders: Marcel GJ Tilanus, Christien EM Voorter, Mathijs Groeneweg
Department of Transplantation Immunology,
Tissue Typing Laboratory,
Maastricht University Medical Center
P.O. Box 5800
6202 AZ Maastricht
Phone: +31 43 3874680
Fax: +31 43 3874678