MACRONUCLEUS GENOME AND TRANSCRIPTOME SEQUENCING AND STRUCTURE ANALYSIS OF EUPLOTES AMIETI
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摘要: 为获得艾美游仆虫(Euplotes amieti)大核基因组结构特征、分析基因功能及其表达调控方式, 研究采用高通量测序技术对艾美游仆虫进行了大核基因组与转录组测序, 结果显示基因组测序最终得到原始reads数据为10.92 Gb, 过滤后得到50287条Contigs。GC含量较低, 为31%; 其中两端同时具有端粒的微染色体数量为27542条, 占54.76%, 只含有一端端粒的基因数量为6118条。Contigs进行基因结构分析, 96.5%的基因能够被预测出功能, 最终得到27650条基因, 平均外显子长度为311.69 bp; 内含子较短, 平均长度为150 bp。转录组测序结果为76219898条, 拼接后获得38588条转录组Unigenes, 其平均长度为1189 bp。将转录组的38588条Unigenes比对发现有2%—3%基因发生了编程性移码, 其中, 绝大多数为+1PRF; 除此之外, 艾美游仆虫的终止密码子还存在重配现象, 其终止密码子为UAA和UAG, 而UGA编码半胱氨酸或硒代半胱氨酸。这与游仆虫属的编程性移码及终止密码子重配的特点一致。将27650条基因组Contigs与38588条转录组Unigenes成功获得注释。基因功能分析显示转录本显著富集于细胞生长与死亡、膜转运、运输与细胞学过程等。将基因组与转录组随机各抽取50个基因进行PCR验证。95%的基因均验证成功。结果表明艾美游仆虫除了具有游仆虫特有的微染色体、密码子重新分配和程序性移码等基因组特征外, 还具有大量“联合微染色体”, 编码大量与感受外界环境变化、细胞周期与蛋白表达调控等相关的特殊蛋白质, 并通过一定量的miRNA与锌指类转录因子对基因进行表达调控。Abstract: To explore genome structure characteristics, gene function and expression regulation of Euplotes amieti, high-throughput sequencing technique was used to sequence the macronucleus genome and transcriptome of Euplotes amieti. A total of 10.92 Gb data and 50287 Contigs were obtained after filtration. The GC content was 31% and the average length of exons and introns were 311.69 bp and 150 bp, respectively. The number of microchromosomes with telomeres at both ends was 27542, accounting for 54.76%, and the number of genes containing only one telomere was 6118. 38588 Unigenes were split, and the average length was 1189 bp. There were 2%—3% PRF in the total Unigenes, and most of which were +1PRF. Besides, the stop codon of Euplotes amieti also has a reconfiguration phenomenon. The stop codons are UAA and UAG, while UGA codes cysteine and selenocysteine. This was consistent with the characteristics of programmed ribosomal frameshifting and stop codon reassignments in Euplotes. 27650 genomic contigs and 38588 transcriptome Unigenes were successfully annotated. Gene function analysis showed that transcripts were significantly enriched in multiple biological processes, mainly involved cell growth and death, membrane transport, transport, and cytology. Moreover, 50 genes were randomly selected from the genome and transcriptome for PCR verification, and 95% of them were successfully verified. The results indicated that in addition to the characteristic microchromosomes and programmed frameshift, the Euplotes amieti had a large number of “combined microchromosomes” and encoded a large number of special proteins related to the perception of changes in the external environment, cell cycle, and protein expression regulation. In addition,, the gene expression may be regulated by some miRNA and zinc finger transcription factors.
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Keywords:
- Euplotes amieti /
- Hronucleus genome /
- Transcriptome /
- Microchromosome /
- Gene annotation
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图 1 基因组及转录组测序结果
A. 基因组注释, B. 转录组注释; GO. GO数据库中被注释到的Conting和Unigene数目; NR. NR数据库中被注释到的Conting和Unigene数目; KEGG. KEGG数据库中被注释到的Conting和Unigene数目; Swissprot. Swissprot数据库中被注释到的Conting和Unigene数目; Pfam. Pfam数据库中注释的Conting数目; Interpro. Interpro 数据库中注释的Conting数目; PFAM. PFAM数据库中注释到的Unigene数目
Figure 1. Genome and transcriptome sequencing results
A. Genome annotation; B. Transcriptome annotation. GO. Number of Contigs and Unigenes annotated in the GO database; NR. Number of Contigs and Unigenes annotated in the NR database; KEGG. Number of Contigs and Unigenes annotated in the KEGG database; Swissprot. Number of Contigs and Unigenes annotated in the Swissprot database; Pfam. Number of Contigs annotated in the Platform database; Interpro. Number of Contigs annotated in the Interpro database; PFAM. Number of Unigenes annotated in the PFAM database
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图 2 基因结构特征比较
A. 平均CDS长度比较; B. 每个基因平均外显子比较结果; C. 几种腹毛类纤毛虫基因结构比较
Figure 2. Comparison of gene structures
A. The average CDS length; B. The average exons of each gene; C. Comparison of the gene structure of some Hypotrichida ciliates
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图 3 艾美游仆虫与多种真核生物基因组比较(进化树基于18S rRNA绘制)
Figure 3. CComparison of representative eukaryotic genome. The tree was constructed based on the sequences of 18S rRNA genes
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图 5 基因组及转录组结果验证
A. 基因组测序1—24号基因验证结果; B. 基因组测序26—50号基因验证结果; C. 转录组测序1—24号基因验证结果; D. 转录组测序26—50号基因验证结果 Marker从上到下依次为2000、1000、750、500、250和100 bp
Figure 5. Genome and transcriptome validation
A. Genome sequencing 1—24 gene validation results; B. Genome sequencing 26—50 gene validation results; C. Transcriptome sequencing 1—24 gene validation results; D. Transcriptome sequencing 26—50 gene validation resultsMarkers from top to bottom are 2000, 1000, 750, 500, 250 and 100 bp
表 1 艾美游仆虫基因组测序数据统计
Table 1 Genomic sequencing data statistics of Euplotes amieti
拼装结果
AssemblerSPAdes
测序软件SPAdes+CAP3拼接软件 最终拼装
结果
Final assembly组装的大小Assembly size (Mb) 95.50 94.56 89.46 片段Contigs (n) 64836 59795 50287 具有两端端粒的片段Number of 2-telomere contigs (n) 27315 27544 27542 具有一端端粒的片段Number of 1-telomere contigs (n) 7719 6537 6118 不具有端粒的片段Number of 0-telomere contigs (n) 29802 25714 16627 长度小于500 bp的片段Number of length<500 bp 25179 22279 13472 GC百分比GC (%) 31 
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表 2 艾美游仆虫转录组拼接序列
Table 2 Transcriptome splicing sequence of Euplotes amieti
序列Sequence 重叠群
Contigs转录组序列
Transcript转录本代表序列Unigene 序列总数Sequence number 68902 60691 38588 序列最大长度Max. length (bp) 16644 16648 16648 序列平均长度Mean length (bp) 609.45 1326.6 1189.99 所有序列长到短排列并相加, 相加的长度达到序列总长度的50%时, 最后一条序列长度 N50 (bp) 1319 1759 1643 长度大于N50 的序列总数
N50 Sequence No.9738 15227 9000 所有序列长到短排列并相加, 相加的长度达到序列总长度的90%时, 最后一条序列长度 N90 (bp) 203 699 581 长度大于N90的序列总数
N90 Sequence No.39290 42291 26558 序列的GC含量GC (%) 33.47 32.99 33.19
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表 3 非编码RNA序列注释
Table 3 Noncoding RNA sequence annotation
类型
Type拷贝数
Copy
(w*)平均长度
Average
length
(bp)总长度
Total length
(bp)占基因组比例Of genome
(%)miRNA 23 125.78 2893 0.003234 tRNA 105 74.32 7804 0.008724 rRNA 28 104.68 2931 0.003276 rRNA 18S 26 104.81 2725 0.003046 28S 2 103 206 0.000230 5.8S 0 0 0 0 5S 0 0 0 0 snRNA 11 164.73 1812 0.002026 snRNA CD-box 1 263 263 0.000294 HACA-box 0 0 0 0 splicing 9 145.22 1307 0.001461 注: *全基因组注释, 综合数据计算平均长度和总长度Note: *Genome annotation, synthesis data calculate average length and total length
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