Pinder Matthew I M, Andersson Björn, Blossom Hannah, Svensson Marie, Rengefors Karin, Töpel Mats
Department of Marine Sciences, University of Gothenburg, Göteborg, Sweden.
NIRAS Sweden AB, Göteborg, Sweden.
Mol Ecol Resour. 2025 May;25(4):e14067. doi: 10.1111/1755-0998.14067. Epub 2025 Feb 4.
Evolutionary changes in populations of microbes, such as microalgae, cannot be traced using conventional metabarcoding loci as they lack intraspecific resolution. Consequently, selection and competition processes among strains of the same species cannot be resolved without elaborate isolation, culturing, and genotyping efforts. Bamboozle, a new bioinformatic tool introduced here, scans the entire genome of a species and identifies allele-rich barcodes that enable direct identification of different genetic strains from a population using amplicon sequencing of a single DNA sample. We demonstrate its usefulness by identifying hypervariable barcoding loci (< 500 bp) from genomic data in two microalgal species, the diploid diatom Skeletonema marinoi and the haploid chlorophyte Chlamydomonas reinhardtii. Across the two genomes, four and twenty-two loci, respectively, were identified that could in silico resolve all analysed genotypes. All of the identified loci are within protein-coding genes with various metabolic functions. Single nucleotide polymorphisms (SNPs) provided the most reliable genetic markers, and among 54 strains of S. marinoi, three 500 bp loci contained, on average, 46 SNPs, 103 strain-specific alleles, and displayed 100% heterozygosity. This high level of heterozygosity was identified as a novel opportunity to improve strain quantification and detect false positive artefacts during denoising of amplicon sequences. Finally, we illustrate how metabarcoding of a single genetic locus can be used to track abundances of S. marinoi strains in an artificial selection experiment. As future genomic datasets become available and DNA sequencing technologies develop, Bamboozle has flexible user settings enabling optimal barcodes to be designed for other species and applications.
微生物种群（如微藻）的进化变化无法通过传统的宏条形码基因座进行追踪，因为这些基因座缺乏种内分辨率。因此，若不进行精细的分离、培养和基因分型工作，就无法解析同一物种菌株之间的选择和竞争过程。本文介绍的一种新的生物信息学工具Bamboozle，可扫描一个物种的整个基因组，并识别富含等位基因的条形码，从而能够通过对单个DNA样本进行扩增子测序，直接从种群中鉴定出不同的遗传菌株。我们通过从两种微藻物种（二倍体硅藻海链藻和单倍体绿藻莱茵衣藻）的基因组数据中识别高变条形码基因座（<500 bp），证明了其有效性。在这两个基因组中，分别鉴定出了四个和二十二个基因座，这些基因座能够在计算机上解析所有分析的基因型。所有鉴定出的基因座都位于具有各种代谢功能的蛋白质编码基因内。单核苷酸多态性（SNP）提供了最可靠的遗传标记，在54株海链藻中，三个500 bp的基因座平均包含46个SNP、103个菌株特异性等位基因，并且显示出100%的杂合性。这种高水平的杂合性被确定为一个新的机会，可用于改进菌株定量，并在扩增子序列去噪过程中检测假阳性假象。最后，我们说明了如何利用单个基因座的宏条形码来追踪人工选择实验中海链藻菌株的丰度。随着未来基因组数据集的可得性以及DNA测序技术的发展，Bamboozle具有灵活的用户设置，能够为其他物种和应用设计最佳条形码。
