College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
Parasit Vectors. 2021 Sep 27;14(1):502. doi: 10.1186/s13071-021-05015-7.
Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix.
Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied.
SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2.
We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control.
柔嫩艾美耳球虫是一种致病性很强的寄生虫，可导致鸡的高死亡率。尽管其基因组序列已经公布，但它的 mRNA 转录本的序列和完整结构仍不清楚，限制了对柔嫩艾美耳球虫中新的生物标志物、药物靶点和遗传功能的探索。
使用 Percoll 密度梯度法收集柔嫩艾美耳球虫第二代裂殖子（MZ-2），并从中提取高质量的 RNA。将单分子实时（SMRT）测序和 Illumina 测序相结合，生成 MZ-2 的转录本。结合我们之前研究中收集的孢子囊（SZ）的 SMRT 测序数据，研究了柔嫩艾美耳球虫的转录组和转录本结构。
SMRT 测序在 MZ-2 中产生了 21923 个一致的同型物。成功鉴定了 17151 种已知基因的新同型物和 3918 种新基因的同型物。我们还鉴定了 2752 个（SZ）和 3255 个（MZ-2）选择性剪接（AS）事件、1705 个（SZ）和 1874 个（MZ-2）具有可变多聚腺苷酸化（APA）位点的基因、4019 个（SZ）和 2588 个（MZ-2）融合转录本、159 个（SZ）和 84 个（MZ-2）假定转录因子（TF）和 3581 个（SZ）和 2039 个（MZ-2）长非编码 RNA（lncRNA）。为了验证融合转录本，对 16 个候选物进行了逆转录-PCR，准确率高达 87.5%。PCR 产物的 Sanger 测序进一步证实了嵌合转录本的真实性。转录本结构的比较分析共在 SZ 和 MZ-2 中鉴定出 3710 个一致的同型物、815 个 AS 事件、1139 个具有 APA 位点的基因、20 个假定的 TF 和 352 个 lncRNA。
我们从柔嫩艾美耳球虫的 SZ 和 MZ-2 中获得了许多长读长同型物，从中鉴定出一系列 lncRNA、AS 事件、APA 事件和融合转录本。TF 信息将提高对转录调控的理解，而融合事件数据将极大地改进柔嫩艾美耳球虫基因模型的草案版本。这些信息为研究柔嫩艾美耳球虫的发育机制提供了线索，并将有助于开发新的球虫病控制策略。
