Abstract
BackgroundEimeria 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.MethodsSecond-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.ResultsSMRT 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.ConclusionsWe 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.Graphical
Highlights
Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens
All polished consensus reads were corrected using the approximately 70 million Illumina cleaned reads as input data (Additional file 7: Table S3), and a total of 192,045 corrected polished consensus reads were obtained from the MZ-2 library (Additional file 8: Table S4)
Based on Single-molecule real-time (SMRT) RNA sequencing, we identified a large number of long-read isoforms in SZ and MZ-2 of E. necatrix and predicted that long non-coding RNAs (lncRNAs), alternative splicing (AS) and alternative polyadenylation (APA) events may be involved in stage differentiation
Summary
Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Eimeria necatrix is a highly pathogenic pathogen that mainly colonizes the midsegments of the small intestine, causing weight loss, poor feed conversion and high mortality [9]. This parasite undergoes a complex life-cycle that includes an exogenous phase (sporogony) and an endogenous phase (schizogony and gametogony) [10]. Second-generation schizonts of E. necatrix, which parasitize the lamina propria, with each schizont producing > 150 secondgeneration merozoites (MZ-2), are considered to be the most concerning stage in terms of pathogenicity due to the severe damage they cause to intestinal tissue [12, 13]. Information on RNA sequences is crucial for understanding the E. necatrix transcriptome and evaluating the structure of genes associated with stage differentiation
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