Laboratory testing

HLA typing using Illumina NGS

We offer a HLA typing service for high-throughput projects. We have capacity to type large cohorts of samples to unambiguous (majority of cases) high resolution (min 4 digit resolution) in only a single pass.

We offer:

  • Class 1 HLA A (Exon 2,3)
  • B (Exon 2,3)
  • C (Exon 2,3) and Class II DRB1,3,4,5 (Exon 2)
  • DQB1 (Exon 2,3)
  • DQA1 (Exon 2)
  • DPB1 (Exon2)

exon specific typing that resolves Alleles to 4 or more digit typing levels covering coding region allelic variation. This can be further combined with other host specific genotyping as required at little extra cost. Our current cost for basic HLA typing in a commercial setting for small numbers of samples (<=50) is now $320 USD per sample that covers all the loci mentioned. For research and academic projects and for collaborative projects the discounts are significant. For further details and to get your project assessed for collaborative gain, and discounts please inquire. Discounts may be available for larger sample sets. There would be little if any discounts applied for only specific subset (Class I or Class II typing alone) unless very large sample numbers need to be processed. For population based HLA linkages sample numbers need to be carefully considered to get statistical significance in most study types. We are happy to advise in any given situation.

The nature of genetic testing and genetic variability may mean that in rare cases we may be unable to identify an Allele at a particular locus.

For pre-configured plates with ideal DNA concentrations in a first pass typing the turnaround times for <1000 samples are approximately 4 weeks from sample arrival. We recommend the following plates Axygen full or half skirted plates (Cat # PCR-96M2-HS-C or PCR-96-FS-C which are compatible with strip caps PCR-02CP-C). Plates must be sealed to prevent cross well contamination during shipping. Please leave wells H6 G12 and H12 empty  and fill order should be A1, B1, C1...etc if possible for us to add controls and avoid transpositions. We ask for ideally 50ul of 50ng/ul DNA with an A260/A280 of at least 1.7 and a size average of >12KB.  Lower amounts and quality can be processed but we cannot guarantee results due to possible allele dropout.

Alternative plates are acceptable please contact us at specimenreception@iiid.com.au if you wish to use alternative plates and layouts for fill order and control wells.  Failure to comply with the specific layout requirements may delay results.

We type Human Leucocyte Antigen (HLA) by either Illumina or Sanger DNA sequencing and we require 50µl of 25 ng/µ l of high quality (A260/A280>=1.7) to perform the full test which involves a proprietary set of 12 PCR reactions covering the major variable exons of the loci we type.  Please note if other typings are required e.g. for ERAP or KIR additional material will be required

We are also able to type a subset of the MAFA LA's (Crab Eating Macaque). Please enquire.

The gene DRB1 is expressed at a level five times higher than its paralogues DRB3, DRB4 and DRB5. DRB1 is present in all individuals. Allelic variants of DRB1 may be linked with DRB3, DRB4 and DRB5. ‘Calls’ in the DRB1 DRB3 DRB4 and DRB5 columns can be confusing.  For some DRB1 alleles different combinations of 3, 4, 5, are expected.  When the assignments in 3, 4, 5 are not detected the notation NP (Not Present) is used. Occasionally a 3, 4, or 5 is expected but not detected, this is still reported as NP but a comment is generally provided (usually as a superscript indicator) denoting expected but not found.

G groups are used for Reporting of Ambiguous Allele Typing (IMGT Ambiguous Alleles http://www.ebi.ac.uk/ipd/imgt/hla/ambig.html).

HLA alleles that have identical nucleotide sequences across the exons encode the peptide binding domains (exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles). They are designated by an upper case ‘G' which follows the first 3 fields of the allele designation of the lowest numbered allele in the group.

However, when typing HLA-DQB1 we can-not use the HLA-DQB G groups since we sequence exon 2 and exon 3. Although we are also sequencing exon 3, there is still ambiguity. We have chosen to make up our own groups, because allele pairs that include two sets of reads that lead to ambiguous calls can result in a large set of possible combinations. We have found when checking through these lists that involve ambiguity that it is difficult.

Hence we define iGroups as:

HLA-DQB1 alleles that have identical nucleotide sequences across the portions of exon 2 and exon 3 that are sequenced for typing, or that are identical across the portion of exon 2 that is sequenced where one or both alleles does not have reference data in the IMGT database for exon 3. Alleles in the same group will be designated by a lower case ‘i’ which follows the first 3 fields of the allele designation of the lowest numbered allele in the group.

As an example a reported iGroup of HLA-DQB1*05:03:01i encompasses all possible allele combinations of HLA-DQB1*05:03:01:01 HLA-DQB1*05:03:01:02 HLA-DQB1*05:10 as defined in the document below

HLA-DRB exon 2 sequences are partially truncated at the 5' end and therefor lead to ambiguous alleles that can result in a large set of possible combinations which makes DRB1/3/4/5 associations difficult to interpret. Hence, iGroups are used.

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