Supplementary Material for: Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the <b><i>ACKR1</i></b> Gene Encoding the Duffy Antigens

<b><i>Background:</i></b> In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of <i>ACKR1</i>, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. <b><i>Materials and Methods:</i></b> After long-range PCR amplification spanning the whole <i>ACKR1</i> gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. <b><i>Results:</i></b> High-quality sequencing reads were obtained for the 162 alleles (accuracy &gt;0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 <i>ACKR1</i>*<i>01</i>, 63 <i>ACKR1</i>*<i>02</i>, and 53 <i>ACKR1</i>*<i>02N.01</i> alleles, respectively. <b><i>Discussion:</i></b> Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

背景：在血型基因组学的崭新时代，对血型基因的参考基因/等位基因序列进行（重新）定义，已成为诊断与研究领域均需达成的重要目标。鉴于新型高性能测序技术已投入应用，本研究拟采用长读长测序（long-read sequencing）技术，在单倍型层面探究编码临床相关达菲抗原（Duffy antigens）的*ACKR1*基因的三种最常见等位基因所存在的变异特征。
材料与方法：针对覆盖整个*ACKR1*基因座（约2.5千碱基对）的区域开展长距离PCR扩增后，本研究利用太平洋生物科学（Pacific Biosciences, PacBio）单分子实时（single molecule, real-time, SMRT）测序技术，对81份已知基因型样本所产生的扩增子进行单读长测序。
结果：本研究成功获取162个等位基因的高质量测序读段，测序准确率＞0.999。通过比对公共数据库，共鉴定出22处核苷酸变异，据此定义出19种单倍型：在46个*ACKR1* *01*、63个*ACKR1* *02*以及53个*ACKR1* *02N.01*等位基因中，分别对应4种、8种及7种单倍型。
讨论：综上，本研究通过第三代测序（即长读长测序）技术定义了一组参考等位基因。该技术可对目标基因座（数千碱基对）实现全景式覆盖，且可作为第二代短读长下一代测序技术的有效补充，在解析新型、罕见及无效等位基因方面具备关键应用价值。

搜集汇总

背景与挑战

背景概述

该数据集是2019年发布的补充材料，聚焦于通过长读长测序技术定义ACKR1基因（编码达菲抗原）的参考等位基因。研究对81个已知基因型样本进行全基因座测序，识别出22个核苷酸变异并定义了19个单倍型，证明长读长测序在血型基因等位基因解析中的关键作用。