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High resolution HLA typing from second generation sequencing data

Cooper, Donald (2015) High resolution HLA typing from second generation sequencing data. PhD thesis, Murdoch University.

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Class I and II genes from the major histocompatibility complex are routinely typed as a screening prior to solid organ and haematopoietic stem cell transplants, and have been linked to susceptibility to many diseases and hypersensitivity reactions to some drugs. Sequenced based typing is the definitive means of typing a person's HLA alleles to high resolution. The development of second generation sequencers beginning with the GS-FLX from Life Sciences, provided the impetus to develop HLA allele callers to deal with this new data. For this thesis, two HLA allele callers were developed, the first based on the expected error pattern for the GS-FLX sequencer and the second based on clustering.

The high cost of a sequencing run can be shared by multiplexing many samples together. This thesis examined GS-FLX sequencing data for the exons encoding the binding pocket at pooling rates of 768 samples per run of Class I genes and 384 samples per run of Class I and II genes. At these high rates of pooling, the sequencing cost per sample is low, but there is a risk that some samples may not receive adequate coverage.

Almost all discordant calls occurred at low minimum coverage, and 99% concordance was achieved when the minimum coverage exceeded three. Overall concordance at all levels of coverage exceeded 90% for the samples with Class I and II data. None of the “High" confidence calls made by either allele caller, were discordant due to call errors.

Analysis of 23 samples sequenced on a single MiSeq run, suggests that this platform has the potential to outperform the GS-FLX as a sequencing platform for HLA-typing.

The clustering algorithm also showed great promise as a tool for resolving mixtures of mitochondrial DNA samples at levels below those achieved by other techniques reported in the literature.

Publication Type: Thesis (PhD)
Murdoch Affiliation: Institute for Immunology and Infectious Diseases
Supervisor: Watson, Mark
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