利用第三代纳米孔长读段测序技术构建和注释蜜蜂球囊菌的全长转录组

doi:10.3864/j.issn.0578-1752.2021.04.017

Abstract

Abstract:

【Objective】Purified mycelia sample (Aam) and spore sample (Aas) were sequenced using third-generation nanopore long-read sequencing technology, followed by construction and annotation of high-quality full-length transcriptome.【Method】Aam and Aas were respectively sequenced using Oxford Nanopore PromethION platform. Guppy software was used to conduct base calling of raw reads. Clean reads were obtained after filtering out short fragments and low-quality raw reads. Full-length transcripts were identified by recognizing primers at both ends of clean reads. Full-length transcripts were aligned to Nr, Swissprot, KOG, eggNOG, Pfam, GO and KEGG databases to gain corresponding annotations. Four approaches such as CPC, CNCI, CPAT, and Pfam were used to predict lncRNAs, and the intersection was deemed to be high-reliability lncRNAs.【Result】In total, 6 321 704 and 6 259 727 raw reads were yielded from nanopore sequencing of Aam and Aas, and after quality control, 5 669 436 and 6 233 159 clean reads were obtained, including 4 497 102 (79.32%) and 4 963 101 (79.62%) full-length clean reads. Additionally, 9 859 and 16 795 non-redundant full-length transcripts were identified, with a N50 of 1 482 and 1 658 bp, an average length of 1 187 and 1 303 bp, and a maximum length of 6 472 and 6 815 bp, respectively. Venn analysis showed that 6 512 non-redundant full-length transcripts were shared by Aam and Aas, while 3 347 and 10 283 ones were specific for Aam and Aas, respectively. Besides, a total of 20 142 full-length transcripts were identified in Aam and Aas, among them 20 809, 11 151, 17 723, 12 164, 11 340 and 9 833 full-length transcripts could be annotated to Nr, KOG, eggNOG, Pfam, GO and KEGG databases, respectively. Most of full-length transcripts were annotated to A. apis, Polytolypa hystricis and Histoplasma capsulatum. Moreover, GO database annotation demonstrated that the above-mentioned full-length transcripts could be annotated to 45 functional terms, involving in cell component-associated terms such as cell part, cell and organelle; molecular function-associated terms such as catalytic activity, binding and transporter activity; and biological process-associated terms such as cellular processes, metabolic processes and single-organism processes. KEGG database annotation indicated that these full-length transcripts could be annotated to 49 pathways, including biosynthesis of antibiotics, ribosome, biosynthesis of amino acid, carbon metabolism, spliceosome and so on. In addition, 648 lncRNAs were identified, including 480 long intergenic RNAs (lincRNAs), 119 anti-sense lncRNAs and 49 sense lncRNAs. 【Conclusion】The first high-quality full-length transcriptome was constructed and annotated in this work, which offers a key basis for exploration of the complexity of A. apis transcriptome, improvement of sequence and functional annotation of reference genome and further study on isoforms’ function of A. apis.

Key words: third-generation high-throughput sequencing technology, nanopore sequencing, full-length transcript, reference transcriptome, honeybee, Ascosphaera apis

DU Yu,ZHU ZhiWei,WANG Jie,WANG XiuNa,JIANG HaiBin,FAN YuanChan,FAN XiaoXue,CHEN HuaZhi,LONG Qi,CAI ZongBing,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,CHEN DaFu,GUO Rui. Construction and Annotation of Ascosphaera apis Full-Length Transcriptome Utilizing Nanopore Third-Generation Long-Read Sequencing Technology[J].Scientia Agricultura Sinica, 2021, 54(4): 864-876.

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References 44

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样品 Sample	原始读段数 Number of raw reads	碱基数 Number of bases	居中长度 N50 (bp)	平均长度 Mean length (bp)	最大长度 Maximum length (bp)	平均质量值 Mean Q score
球囊菌菌丝Aam	6 321 704	6 271 320 854	1 094	992	9 421	Q10
球囊菌孢子Aas	6 259 727	6 553 996 867	1 157	1 047	13 060	Q10

样品 Sample	全长转录本数 Number of full-length transcripts	碱基数 Number of bases	居中长度 N50 (bp)	平均长度 Mean length (bp)	最大长度 Maximum length (bp)
球囊菌菌丝Aam	9 859	11 706 153	1 482	1 187	6 472
球囊菌孢子Aas	16 795	21 899 133	1 658	1 303	6 815

Construction and Annotation of Ascosphaera apis Full-Length Transcriptome Utilizing Nanopore Third-Generation Long-Read Sequencing Technology

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样品 Sample	有效读段数 Number of clean reads	全长有效读段数 Number of full-length clean reads	全长有效读段数的占比 Percentage of full-length clean reads (%)
球囊菌菌丝Aam	5 669 436	4 497 102	79.32
球囊菌孢子Aas	6 233 159	4 963 101	79.62

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