Warren René L, Birol Inanc
BC Cancer Genome Sciences Centre, Vancouver, BC, Canada.
Department of Medical Genetics, University of British Columbia, Columbia, BC, Canada.
Curr Protoc. 2025 Mar;5(3):e70124. doi: 10.1002/cpz1.70124.
Long-read sequencing platforms such as the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms now offer sufficient read lengths, throughput, and accuracy at competitive costs to analyze polymorphic regions of the human genome, including the highly complex human leukocyte antigen (HLA) gene cluster-a cornerstone of human immunity. Here, we present a streamlined protocol for predicting HLA signatures from whole-genome shotgun (WGS) long-read sequencing data by directly streaming sequence alignments into HLAminer. This method is as simple as running minimap2, scales efficiently with the number of sequences, and works with any read aligner compatible with the SAM file format-eliminating the need to store bulky alignment files on disk. We provide a step-by-step guide for predicting HLA class I and class II alleles from third-generation long-read sequencing data and demonstrate the robustness of predictions even with older, less accurate WGS nanopore datasets and relatively low (10×) sequencing coverage. Code availability: HLAminer is available under the BC Cancer software license agreement (academic use) at https://github.com/bcgsc/HLAminer. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: HLA prediction from streamed ONT or PacBio long-read alignments.
牛津纳米孔技术公司（ONT）和太平洋生物科学公司（PacBio）等长读长测序平台，如今在成本具有竞争力的情况下，提供了足够的读长、通量和准确性，可用于分析人类基因组的多态性区域，包括高度复杂的人类白细胞抗原（HLA）基因簇——人类免疫的基石。在此，我们展示了一种简化方案，可通过将序列比对直接流式传输到HLAminer中，从全基因组鸟枪法（WGS）长读长测序数据预测HLA特征。该方法与运行minimap2一样简单，能随着序列数量高效扩展，并且适用于任何与SAM文件格式兼容的读段比对工具，无需在磁盘上存储庞大的比对文件。我们提供了一份从第三代长读长测序数据预测HLA I类和II类等位基因的分步指南，并证明即使使用较旧、准确性较低的WGS纳米孔数据集以及相对较低（10×）的测序覆盖度，预测结果依然稳健。代码可用性：HLAminer可根据BC癌症软件许可协议（学术用途）在https://github.com/bcgsc/HLAminer获取。© 2025作者。由威利期刊有限责任公司出版的《当前协议》。基本方案：从流式传输的ONT或PacBio长读长比对中进行HLA预测。
