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We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS.

Aim: A ‘Silver Standard’ for HLA data collection and reporting has been described at ImmPort (immport.niaid.nih.gov, “Proposal for HLA Data Validation”) to address ambiguity resolution in the recording and reporting of HLA typing results. While standards are critical for HLA data interoperability, they are not meaningful until useful tools are developed and made available for community use. We are developing distributable tools that implement this silver standard. Here we describe the development a web service to create, update, and retrieve HLA typing data in standardized formats without the need for NMDP allele codes and the corresponding inherent introduction of new ambiguities. Methods: ReST web services with HTTP negotiation are being developed employing a Java library that manages HLA typing data using standardized formats. These formats include the XML based Histoimmunogenetics Markup Language (HML) and a simple character-delimited string format (GL String) able to encode ambiguity within HLA typing. Resources are identified with a simple Uniform Resource Identifier (URI). Results: The services build on a foundation of an open access database schema for IMGT/HLA reference sequence data (updated quarterly), and objects such as alleles, lists of alleles, haplotypes, genotypes, lists of genotypes and multi-locus unphased genotypes. Public services include creating, updating, and retrieving these objects. Content negotiation allows data retrieval in a variety of formats including GL String, HML, HTML, JSON, and QR Code. Conclusions: The tools being developed here provide the HLA researcher, clinician and lab technician a common resource for managing HLA data in a standardized way. We envision these tools to augment workflows through creating new instances of HLA typing objects when needed, and retrieval of those objects and their associated metadata when called upon. [Copyright &y& Elsevier]

Aim: We previously showed that Shannon’s entropy could be successfully used to quantify the typing resolution of HLA data in stem cell registries. Here, we present an improvement of this measure, termed typing ambiguity score, which enables direct and systematic comparison across methods, data sets and populations. Methods: Typing ambiguity score is calculated from normalized Shannon’s entropy. It ranges between 0 and 1, where 1 is given to a typing with no ambiguity and 0 to a typing with maximum ambiguity. We differentiate between ambiguity in (phased) HLA genotypes and ambiguity in un-phased genotypes, which has a more practical application for donor and patient matching. Furthermore, we implement a web-based typing ambiguity assessment tool, integrated with an existing application called HaploStats that facilitates access to HLA haplotype frequencies. Results: Maximum ambiguity is obtained for a typing that results in equally probable set of outcomes, since for such cases we have the least information on what the true outcome is. For example, outcome with probabilities p = (0.5, 0.5) has a score equal to 1, while outcome with probabilities p = (0.95, 0.05) has a score of 0.28. To demonstrate how this score can be used to evaluate typings in a stem cell registry, we compute overall and per-locus ambiguity scores for HLA typings in the BeTheMatch® (US) registry, and explore how it varies with data resolution, typing method, time, and population. Conclusions: We present a method to objectively quantify and compare ambiguity in HLA typing data obtained via different methods. An analysis of HLA typing data in the US registry demonstrates the utility of this method for stem cell registries and HLA typing labs. We show that the typing ambiguity score can compare typing methodologies, determine which methods are best suited for which populations, and help define acceptable HLA typing for donor recruitment. [ABSTRACT FROM AUTHOR]

Aim: Using Shannon’s entropy, we objectively measured the HLA typing ambiguity of various commercial Sequence-Specific Oligonucleotide (SSO) protocols (groups of SSO kits used in tandem). We previously compared the HLA typing ambiguity obtained by serology, allele family level DNA-based typing, SSO, and single-pass sequence based typing (SBT). However, commercial companies have made significant advances in SSO technology in recent years, and those results only evaluated early generic versions of SSO kits. Methods: For several US populations, and for 5 HLA loci relevant to transplantation (HLA-A, -B, -C, -DRB1, -DQB1), we identify a set of commonly used SSO protocols. We use population-level haplotype frequency data to generate a cohort of simulated subjects, followed by 5-locus genotype imputation to generate a list of genotypes that would result from SSO typing each simulated subject. The imputation step computes the relative likelihood of each genotype, which is then used for entropy calculation. Results: Distribution of entropies across populations and for each locus is generally similar, while the magnitudes differ. For example, entropy for locus HLA-A in African American population reaches 0.19, while the maximum entropy in Caucasians is 0.055. We generally observed that protocols comprised of newer kits had lower ambiguity across all populations. In certain populations, some newer kits had higher entropy than older ones. This may indicate those kits were designed to detect alleles uncommonly found in those populations, but resolving ambiguity in other populations. Conclusions: Ambiguity in SSO typing kits has reduced substantially since their introduction in the mid-1990s. Results also indicate a European focus toward selection of additional probes, which is suboptimal for typing diverse populations. We hope to use Shannon’s entropy next to evaluate group-specific sequence primer strategies with SBT. These results will guide selection of HLA typing methods by researchers and laboratories. [Copyright &y& Elsevier]

Aim: The IMGT/HLA Database provides a specialist database for sequences of the human major histocompatibility complex, known as HLA and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The database currently provides exports of the data in a variety of formats; this is been expanded to include Extensible Markup Language (XML). Methods: The XML format defines a set of rules for encoding documents in a format that is both human and machine-readable. XML is becoming a common format for exporting database contents for dissemination over the World Wide Web. A collaborative working group has provided an XML export of the data contained within the IMGT/HLA Database. This export format would be supported by tools for the utilisation of this dataset into different database formats. Results: The XML format combines the data included in the sequence alignments with the data available in the individual allele reports. This combination of data has not previously been available in a single format. The XML format will enable users to identify the regions within the DNA sequence, such as exons, as well as reconstruct the sequence alignments. This machine-readable format provides a standardised format for importing data from the reference database into a variety of programs. In addition the collaborative project has developed a suite of tools for importing the data into different database schema for allowing incorporation into different laboratory systems. Conclusions: The XML files will be regularly updated as part of the quarterly releases of the IMGT/HLA Database. A beta test version of the XML format and associated tools will shortly be available from the hla.alleles.org and the National Marrow Donor Program websites. [Copyright &y& Elsevier]