Sequencing and Analysis of Full-length Transcriptome from Medicago truncatula

doi:10.13560/j.cnki.biotech.bull.1985.2021-0191

Abstract

Abstract:

For deep analysis and exploring of the complete structure of mRNA from Medicago truncatula,a model plant in Leguminosae,the single-molecule long-read sequencing technology（SMRT）was used to sequence and analyze the full-length transcriptome of M. truncatula. A total of 7 728 183 subreads and 509 014 full-length non-chimeric reads（FLNC）were obtained. By comparing the reference genomes of R108 and A17,94.36% and 93.01% isoforms were identified as mapped reads,respectively. A total of 8 406 alternative splicing events was identified and the majority was intron retention（RI）. The 23 926 genes were detected,of 12 049 genes had 295 545 transcripts,in which at least there was one poly（A）site. In addition,3 223 transcription factors,6 595 long non-coding RNAs（lncRNA）and 479 fusion transcripts were identified. The results showed the feasibility of deep sequencing M. truncatula transcriptome by SMRT,which provides data supplement for better utilizing the genome resource of M. truncatula.

Key words: Medicago truncatula, single-molecule long-read sequencing, alternative splice, fusion transcript

SHANG Xiao-yao, ZHOU Ling-fang, YIN Qian-qian, CHAO Yue-hui. Sequencing and Analysis of Full-length Transcriptome from Medicago truncatula[J]. Biotechnology Bulletin, 2021, 37(8): 131-140.

Figures/Tables 13

Table 1 Statistics of sequencing data by SMRT

类别 Category	数目 Amount	最小长度 Min length/bp	最大长度 Max length/bp	平均长度 Mean length/bp	N50长度 N50 length/bp
Subread	7 728 183	51	73 164	1 524	2 051
CCS	659 220	200	17 791	2 257	2 757
FLNC	509 014	200	17 128	2 047	2 440
ICE consensus	243 832	200	17 128	2 305	3 367
Polished consensus	243 676	166	17 128	2 321	3 380
Corrected consensus	236 243	260	17 954	2 425	3 496

Fig. 1 GMAP analysis of corrected reads to reference genome

Fig. 2 Classification of RC61125 sequence

Table 2 Functional annotations of novel genes

数据库 Databases	新基因注释数量 Number of functional annotated novel genes
NR	5 183
SwissProt	2 370
KEGG	4 532
KOG	1 884
GO	1 717
NT	6 916
PFAM	1 717
总计 Total	7 209
未注释 Unannotated ones	227

Fig. 3 Nr homologous species distribution diagram of novel genes

Fig. 4 RT-PCR validation of novel genes 1-5：Randomly selected novel genes. M：DNA marker

Table 3 CDS and UTR identification

序列来源 Sequence origin	编码区数量 Number of CDS	编码区平均长度 Mean CDS length/bp	完整编码区数量 Number of complete CDS	3'非编码区数量 Number of 3'-UTR	5'非编码区数量 Number of 5'-UTR
SMRT	36 235	1 027.03	13 451	1 990	11 071
R108	61 020	874.54	16 313	3 895	4 928

Fig.5 Distribution of exons in transcripts Pac-Bio：SMRT transcripts. R108：Transcripts from M. truncatula R108 reference genome.A17：Transcripts from M. truncatula A17 reference genome

Fig.6 Numbers and types of AS events SE：Skipped exon. MX：mutually exclusive exon. A5：Alternative 5' splice site. A3：Alternative 3' splice site. RI：Retained intron. AF：First exon is alternative splice. AL：Last exon is alternative splice

Table 4 Number of transcripts in SMRT sequence and reference genomes

转录本数目 Number of transcript	SMRT测序 SMRT sequence	参考基因组 Reference genome
1	11 730	52 332
2	4 713	2 308
3	2 660	617
4	1 614	246
5	965	96
6	618	58
7	415	21
8	334	10
9	216	8
10	147	5
>10	514	5

Fig.7 Identification of lncRNA A：Venn diagram of lncRNAs predicted by CPC,CNCI,PLEK and Pfam methods.B：Proportions of 4 types of lncRNA identified by SMRT

Fig. 8 RT-PCR validation of fusion transcripts 1-5：Randomly selected fusion transcripts. M：DNA marker

Fig. 9 Analysis of poly（A）sites A：Distribution of the number of poly（A）sites per gene. B：Nucleotide composition around poly（A）cleavage sites. C：MEME analysis of repeated sequence in the upstream of the poly（A）site. D：MEME analysis of repeated sequence in the downstream of the poly（A）site

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