Project: Full-length HLA allele-specific hemizygous Sanger sequencing

Extension of incomplete HLA allele sequences in the IPD-IMGT/HLA database

Project leaders: Marcel GJ Tilanus, Christien EM Voorter, Mathijs Groeneweg

Detailed project description:

The goal of this component is to extend sequences of as many incompletely covered HLA alleles as possible by full length unambiguous Sanger Sequencing.

All laboratories can participate by including unique samples in this study with HLA alleles, of which the complete genomic sequence is not yet known. It is possible to sequence in your own laboratory or send DNA to the Maastricht laboratory and laboratories are welcome to join the component at any time. 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 laboratories that are not able to perform NGS, this component offers the opportunity to actively participate in the workshop with Sanger Sequencing. Within this component, full-length Sanger sequencing, using the Maastricht SSBT method and reagents, will be performed on alleles with partially known allele sequences, with allele separation based on the available low resolution typing data. Laboratories that would like to perform and submit NGS data are welcome to participate in this workshop as well. Once sequencing in your own laboratory is performed the Maastricht lab may ask for a DNA sample for confirmatory purposes.

The project has 5 different phases:

Phase 1: Submission of alleles available

At the start of phase 1 of the project, participants are requested to submit a list of alleles that they have available in the laboratory, for which:

(1) The full length sequence is not yet known in the IMGT/HLA database, or

(2) No confirmation sequence is available.

It is also necessary to indicate on which technique the assignment of the allele of the submitted sample has been based (e.g. exons 2 and 3 SSP HR, SSO HR, SBT). From the selected samples there should be an HLA typing available of preferably HLA-A, -B, -C, -DRB1, -DQB1 and –DPB1, but at a minimum HLA-A, -B and –DRB1 (minimal first field resolution, preferably higher resolution). Furthermore, material (preferably DNA) must be made available to the organizers in Maastricht.

Phase 2: Selection of alleles     

In phase 2, based upon the submitted data, alleles will be selected by the organizers in Maastricht. Lists of alleles that are addressed will be published on the 18th IHIWS website.

Phase 3: Full-length sequencing

In phase 3, full-length hemizygous Sanger sequencing of the allele according to the Maastricht protocol is performed, either by the participating laboratory itself or by the Maastricht laboratory, after sending the DNA of interest to Maastricht.

An alternative approach is that full-length NGS sequencing is performed in the participating laboratory.  DNA must be made available to the organizers in Maastricht for confirmatory purposes if necessary.

For submission purposes sequencing must be performed (forward and reversed) on two separate PCR products. Again, this can be performed by the participating laboratory or by the organizers in Maastricht.

Phase 4: Analysis and verification of data

Phase 4 encompasses the analysis of the data. If the laboratory has performed the sequencing of the allele, the raw sequence data must be send to Maastricht. The data provided can be e.g. ab1 files for ABI sequencers or fastq files, for any NGS platform. Furthermore, sequencing data in other formats will be considered and can be submitted. Maastricht will evaluate if the data can be analyzed and an analysis strategy will be discussed with the submitting laboratory. All sequence data will be evaluated and verified by the Maastricht lab.

Phase 5: Submission to EMBL-ENA and IPD-IMGT/HLA database

The final phase is the submission of the full-length allele sequences to the EMBL-ENA and IPD-IMGT/HLA database. Preferentially, submission will be performed by the laboratory that has submitted the sample (after verification of the sequence data by the organizers in Maastricht).  EMBL-ENA submissions can be performed by Saddlebags, an automated software tool for the preparation and submission of HLA sequences to the EMBL-ENA database. It is freely available, and can be downloaded from https://github.com/transplantation-immunology-maastricht/saddle-bags. A  software tool for automated submission to IPD-IMGT/HLA using Saddlebags is in preparation.

Milestones in years:

2019: phases 1, 2, 3 and 4

2020: phases 1, 2, 3, 4 and 5

2021: complete the submitted samples, evaluation and final overview .


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