for Acceptance of New Alleles
Strict quality standards for DNA sequencing are essential to prevent naming of large numbers of non-existent alleles. Here are the guidelines for acceptance of new sequences for allele name assignment:
- Where a sequence is obtained from cDNA, or where PCR products are cloned before sequencing, several clones should have been sequenced.
- Where a sequence is derived from cDNA, at least two separate RT-PCR reactions should have been performed.
- If direct sequencing of PCR-amplified material is performed, products from at least two separate PCR reactions should have been sequenced.
- Sequencing should always be performed in both forward and reverse directions.
- In individuals who are heterozygous for a locus and where one of the alleles is novel, the novel allele must be sequenced in isolation from the second allele. Thus an allele sequence, where both alleles of a heterozygous individual are sequenced together, is insufficient evidence for assignment of an official designation.
- Sequence derived solely from the primers used for amplifying an allele should not be included in the submitted sequence.
- Where possible, a novel sequence should be confirmed by means of a DNA typing method, such as PCR-sequence-specific primer (-SSP) or PCR-restriction fragment length polymorphism (-RFLP). Where a new sequence contains a novel mutation, a previously unseen combination of nucleotides (sequence motif), or a few nucleotide differences from another published sequence, it must be confirmed by a DNA typing method for the assignment of an official designation. This may require the use of newly designed primers or a unique set of restriction enzymes to cover the new mutation; these reagents should also be described.
- An accession number in a databank should have been obtained. Sequences may be submitted to the databases online at the following addresses:
- Full-length sequences are preferable, though not essential; the minimum requirements are exons 2 and 3 for an SLA class I sequence and complete exon 2 for an SLA class II sequence.
- Partial-length sequences derived using locus-specific PCR amplification must show evidence that the primers used are specific for the designated locus. There must be evidence from family studies that the primer set used does not amplify more than two alleles in any individual when a single allele is inherited from each parent. Specificity of the primer set used should have been verified from amplification of at least one known, full-length allele of that locus.
- Where possible, a paper in which the new sequence is described should have been submitted for publication.
- Wherever possible, DNA or other material, in particular cell lines, should be made available in a publicly accessible repository or at least in the originating laboratory. Documentation on this will be maintained by the International Society for Animal Genetics (ISAG) SLA Nomenclature Committee and the chair of this Committee will serve as curator of this documentation.
- Submission of a sequence to the ISAG SLA Nomenclature Committee should be performed using the online submission tool available at the IPD-MHC sequence database at http://www.ebi.ac.uk/ipd/mhc/submit.html. Researchers are expected to complete a questionnaire relating to the sequence and provide a comparison of their new sequence with known related alleles. If the sequence cannot be submitted using the online web tools, researchers should contact email@example.com directly for details of alternative submission methods.
For more information on the SLA nomenclature
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