Genome-wide gene expression analysis of amphioxus (Branchiostoma belcheri) following lipopolysaccharide challenge using strand-specific RNA-seq

Abstract

Amphioxus is the closest living proxy for exploring the evolutionary origin of the immune system in vertebrates. To understand the immune responses of amphioxus to lipopolysaccharide (LPS), 5 ribosomal RNA (rRNA)-depleted libraries of amphioxus were constructed, including one control (0 h) library and 4 treatment libraries at 6, 12, 24, and 48 h post-injection (hpi) with LPS. The transcriptome of Branchiostoma belcheri was analyzed using strand-specific RNA sequencing technology (RNA-seq). A total of 6161, 6665, 7969, and 6447 differentially expressed genes (DEGs) were detected at 6, 12, 24, and 48 hpi, respectively, compared with expression levels at 0 h. We identified amphioxus genes active during the acute-phase response to LPS at different time points after stimulation. Moreover, to better visualize the resolution phase of the immune process during immune response, we identified 6057 and 5235 DEGs at 48 hpi by comparing with 6 and 24 hpi, respectively. Through real-time quantitative PCR (qRT-PCR) analysis of 12 selected DEGs, we demonstrated the accuracy of the RNA-seq data in this study. Functional enrichment analysis of DEGs demonstrated that most terms were related to defense and immune responses, disease and infection, cell apoptosis, and metabolism and catalysis. Subsequently, we identified 1330, 485, 670, 911, and 1624 time-specific genes (TSGs) at 0, 6, 12, 24, and 48 hpi. Time-specific terms at each of 5 time points were primarily involved in development, immune signaling, signal transduction, DNA repair and stability, and metabolism and catalysis, respectively. As this is the first study to report the transcriptome of an organism with primitive immunity following LPS challenge at multiple time points, it provides gene expression information for further research into the evolution of immunity in vertebrates.

Characterization of ladybird Henosepilachna vigintioctopunctata transcriptomes across various life stages

“…Summary data for the raw reads generated by the sequencer and the clean reads are listed in Table 2 . Clean reads accounted for 81.78–86.32% of the raw reads in the four libraries, and the percentage of raw reads with Q>20 exceeded 98%, which is better than the results of other similar studies 18 , 20 . These results indicate that the filtering of raw reads was robust.…”

Genome-Wide Identification and Transcriptomic Analysis of MicroRNAs Across Various Amphioxus Organs Using Deep Sequencing

“…Approximately 34 million clean reads for each of the eight organ samples, including skin, ovary, notochord, nerve cord, muscle, intestine, hepatic cecum, and gill, were obtained after filtering low-quality reads and contaminants ( Figures 1A–C and Supplementary File 2 ). All of the organ samples showed peak length of the miRNAs at about 22 to 23 nt ( Supplementary File 3 ), as has been previously described for B. belcheri and other animals (Zhang et al, 2017a). All sequencing data have been released to the NCBI SRA database under accession number SRR9951226-SRR9951233.…”

“…To ensure the accurate discovery of novel miRNAs through verification of the secondary structures, novel miRNA precursors (pre-miRNAs) were analyzed using RNAfold (Höner zu Siederdissen et al, 2011) to estimate whether the secondary structure of the precursor is a perfect stem-loop formation. Furthermore, the predicted novel miRNA tags/sequences with mapped tags of fewer than five were discarded (Zhang et al, 2017a), and the remainder retained as the novel mirDeep2 miRNAs. Meanwhile, the additional prediction software Mireap (Yuan et al, 2013) was used to discover novel miRNAs by predicting the hairpin structure, dicer cleavage site, and minimum free energy of the unannotated sRNA tags that had been mapped to the genome.…”

“…The data shown are presented as the mean ± SD, and figures were generated by SigmaPlot 12.0 (Systat Software Inc., San Jose, CA). According to methods described in our previous studies (Zhang et al, 2017b), consistency of miRNA expression patterns between deep sequencing and quantitative real-time PCR (qRT-PCR) was evaluated by calculating the Pearson correlation coefficient and its significance ( P value) level in IBM SPSS statistics 22 software (International Business Machines Corp., New York, USA).…”

Genome-wide gene expression analysis in the amphioxus,Branchiostoma belcheriafter poly (I: C) challenge using strand-specific RNA-seq

“…qRT-PCR was performed on the ABI 7300 Real-Time PCR System (Applied Biosystems, USA) using SYBR Premix ExTaq (Takara, Japan) using the technological processes and reaction systems used in our previous studies [ 62 ]. EF1A was used as a reference gene, and relative gene expression was calculated using the 2 -∆∆CT method [ 67 , 68 , 69 ]. qRT-PCR reaction of each gene for each assay was included three technical replications and three biological replications, and final normalized results in expression survey of six genes are presented as means ± standard deviation (SD).…”