Molecular Ecology and Evolution Lab, Department of Biology, Lund University, Sölvegatan 37, SE-22362, Sweden.
Molecular Ecology and Evolution Lab, Department of Biology, Lund University, Sölvegatan 37, SE-22362, Sweden.
Int J Parasitol. 2018 Oct;48(12):947-954. doi: 10.1016/j.ijpara.2018.05.009. Epub 2018 Aug 11.
Genomic sequencing of avian haemosporidian parasites (Haemosporida) has been challenging due to excessive contamination from host DNA. In this study, we developed a cost-effective protocol to obtain parasite sequences from naturally infected birds, based on targeted sequence capture and next generation sequencing. With the genomic data of Haemoproteus tartakovskyi as a reference, we successfully sequenced up to 1000 genes from each of the 15 selected samples belonging to nine different cytochrome b lineages, eight of which belong to Haemoproteus and one to Plasmodium. The targeted sequences were enriched to ∼10-fold, and mixed infections were identified as well as the proportions of each mixed lineage. We found that the total number of reads and the proportions of exons sequenced decreased when the parasite lineage became more divergent from the reference genome. For each of the samples, the recovery of sequences from different exons varied with the function and GC content of the exon. From the obtained sequences, we detected within-lineage variation in both mitochondrial and nuclear genes, which may be a result of local adaptation to different host species and environmental conditions. This targeted sequence capture protocol can be applied to a broader range of species and will open a new door for further studies on disease diagnostics and comparative analysis of haemosporidians evolution.
由于宿主 DNA 的过度污染，对禽血孢子虫（Haemosporida）的基因组测序一直具有挑战性。在这项研究中，我们基于靶向序列捕获和下一代测序，开发了一种从自然感染的鸟类中获得寄生虫序列的经济有效的方案。利用 Haemoproteus tartakovskyi 的基因组数据作为参考，我们成功地从属于九个不同细胞色素 b 谱系的 15 个选定样本中的每个样本中测序了多达 1000 个基因，其中 8 个属于 Haemoproteus，1 个属于 Plasmodium。靶向序列的富集倍数约为 10 倍，还鉴定了混合感染以及每种混合谱系的比例。我们发现，当寄生虫谱系与参考基因组的差异越大时，读取总数和测序外显子的比例就越低。对于每个样本，来自不同外显子的序列的回收率随外显子的功能和 GC 含量而变化。从获得的序列中，我们检测到了线粒体和核基因中的种内变异，这可能是寄生虫对不同宿主物种和环境条件的局部适应的结果。这种靶向序列捕获方案可应用于更广泛的物种，并为疾病诊断和血孢子虫进化的比较分析的进一步研究开辟新的途径。
