Prevalence of ambiguous allele combinations in sequence-based typing

Adams, Sharon; Barracchini, Kathleen C.; Chen, Deborah; Robbins, Fu-Meei; Stroncek, David F.; Marincola, Francesco M.

doi:10.1016/j.humimm.2003.08.153

Cited by 1 publication

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“…In addition, recent advances made possible to perform SBT at a high throughput level in routine HLA typing laboratories [4]. The biggest challenge that SBT of HLA alleles incurs is the resolution of intrinsic cis/trans ambiguities that cannot be solved by SBT unless time consuming cloning of individual genes is performed [6]. This is because nucleotide incorporation proceeds simultaneously along all DNA templates in a SBT reaction [7].…”

Section: Introductionmentioning

confidence: 99%

Pyrosequencing™ : A one-step method for high resolution HLA typing

Ramón¹,

Braden²,

Adams

et al. 2003

J Transl Med

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While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.

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Section: Introductionmentioning

confidence: 99%