De Novo RNA Seq Assembly and Annotation of Trigonella foenum-graecum L. (SRR066197)  

Sagar Patel1 , Dipti B. Shah1 , Hetalkumar J. Panchal2
1.G. H. Patel Post Graduate Department of Computer Science and Technology, Sardar Patel University, Vallabh Vidyanagar, Gujarat-388120, India 2.Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, Gujarat-395007, India
Author    Correspondence author
Legume Genomics and Genetics, 2014, Vol. 5, No. 7   doi: 10.5376/lgg.2014.05.0007
Received: 23 Oct., 2014    Accepted: 18 Dec., 2014    Published: 24 Dec., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Patel et al., 2014, De Novo RNA Seq Assembly and Annotation of Trigonella foenum-graecum L. (SRR066197), Legume Genomics and Genetics, Vol.5, No.7 1-7 (doi: 10.5376/lgg.2014.05.0007)

Abstract

Trigonella foenum-graecum commonly known as fenugreek, is a small seeded annual dicotyledonous legume belonging to the subfamily Fabaceae, family Leguminosae. Different parts of the plant such as leaves and seeds are consumed in India. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the Transcriptome. This study is focus on RNA-seq of Trigonella foenum-graecum L. of SRR066197 from NCBI database for de novo Transcriptome analysis. A total of 627,117 million single reads were generated with N50 of 470 bp. Sequence assembly contained total 7256 contigs which is further search with known proteins, a total of 2258 genes were identified. Among these, only 192 unigenes were annotated with 10179 gene ontology (GO) functional categories and sequences mapped to 87 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). These data will be useful for gene discovery and functional studies and the large number of transcripts reported in the current study will serve as a valuable genetic resource of the Trigonella foenum-graecum L..

Keywords
Transcriptome; Bioinformatics; Trigonella foenum-graecum L.

Trigonella foenum-graecum L. is an annual, leguminous plant. It has tri-foliate, obovate and toothed, light green leaves. Its stems are erect, long and tender. Blooming period occurs during summer. Flowers are yellow-white, occurring singly or in pairs at the leaf axils. Fruit is a curved seed-pod, with ten to twenty flat and hard, yellowish-brown seeds. They are angular- rhomboid, oblong or even cubic, and have a deep furrow dividing them into two unequal lobes.

One of the main uses of this plant is as Medicinal use like, It has been used for centuries for different female conditions, brain and nervous system ailments, skin, liver and metabolic disorders. It is also considered highly beneficial for respiratory and gastrointestinal problems.
It is a highly potent female herb, since it helps relaxing the uterus and relieving menstrual pains, and is an excellent stimulator of milk production in nursing mothers; for the gastrointestinal tract, Trigonella foenum-graecum L.is usually suggested in treatments of poor digestion, gastric inflammations, enteritis, especially for convalescents. It can also be used in cases of weight loss, poor appetite and even in treatment of anorexia nervosa. Different blood conditions, such as anemia, and nervous system disorders (neurasthenia) can also be successfully treated with Trigonella foenum-graecum L. and for the respiratory conditions, Trigonella foenum-graecum L. is excellent in treatment of bronchitis, mucous congestions, different infections, tuberculosis.
Next generation sequencing methods for high throughput RNA sequencing (transcriptome) is becoming increasingly utilized as the technology of choice to detect and quantify known and novel transcripts in plants. This Transcriptome analysis method is fast and simple because it does not require cloning of the cDNAs. Direct sequencing of these cDNAs can generate short reads at an extraordinary depth. After sequencing, the resulting reads can be assembled into a genome-scale transcription profile. It is a more comprehensive and efficient way to measure Transcriptome composition, obtain RNA expression patterns, and discovers new exons and genes (Mortazavi et al., 2008; Wang et al.,2009); sequencing data of Transcriptome was assembled using various assembly tools, functional annotation of genes and pathway analysis carried with various Bioinformatics tools. The large number of transcripts reported in the current study will serve as a valuable genetic resource for Trigonella foenum-graecum L.
High-throughput short-read sequencing is one of the latest sequencing technologies to be released to the genomics community. For example, on average a single run on the Illumina Genome Analyser can result in over 30 to 40 million single-end (~35 nt) sequences. However, the resulting output can easily overwhelm genomic analysis systems designed for the length of traditional Sanger sequencing, or even the smaller volumes of data resulting from 454 (Roche) sequencing technology. Typically, the initial use of short-read sequencing was confined to matching data from genomes that were nearly identical to the reference genome. Transcriptome analysis on a global gene expression level is an ideal application of short-read sequencing. Traditionally such analysis involved complementary DNA (cDNA) library construction, Sanger sequencing of ESTs, and microarray analysis. Next generation sequencing has become a feasible method for increasing sequencing depth and coverage while reducing time and cost compared to the traditional Sanger method (L J Collins et al., 2008).
1 Methods
1.1 Sequence Retrieval:
This study is focus on the de novo assembly and sequence annotation of Trigonella foenum-graecum L. of SRR066197 from NCBI database. Raw data downloaded from NCBI SRA (http://trace.ncbi.nlm. nih.gov/Traces/sra/?run= SRR066197) which is from LS454 platform- 454 GS FLX and the sample is single ended with 627,117 spots and 45.2% GC content. Raw sequence was converted in to fasta file format for further annotation by using SRA TOOL KIT from NCBI. (http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi? view=software)
1.2 NGS QC Toolkit
It is an application for quality check and filtering of high-quality data. This toolkit is a standalone and open source application freely available at http://www.nipgr.res.in/ngsqctoolkit.html. The toolkit is comprised of user-friendly tools for QC of sequencing data generated using Roche 454 and Illumina platforms, and additional tools to aid QC (sequence format converter and trimming tools) and analysis (statistics tools). A variety of options have been provided to facilitate the QC at user-defined parameters. The toolkit is expected to be very useful for the QC of NGS data to facilitate better downstream analysis (Patel RK, et al, 2011).
1.3 De novo sequence assembly by CLC GENOMICS WORKBENCH 7
A comprehensive and user-friendly analysis package for analyzing, comparing, and visualizing next generation sequencing data. This package was used for de novo sequence assembly of sequence with by default parameters of de novo assembly tool (http://www.clcbio.com/products/clc-genomics-workbench/).
1.4 BLASTX
The assembled file was further considered for annotation in which first step was to identify translated protein sequences from contigs. BLASTX at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch &LINK_LOC=blasthome) performed with changing few parameters like non redundant protein database (nr) selected as Database; Eudicots selected in organism option and in Algorithm parameters Max target Sequences set to 10 and Expect threshold set to 6.
1.5 Blast2GO
Blast2GO is an ALL in ONE tool for functional annotation of (novel) sequences and the analysis of annotation data (http://www.blast2go.com/b2ghome). Based on the results of the protein database annotation, Blast2GO was employed to obtain the functional classification of the unigenes based on GO terms. The transcript contigs were classified under three GO terms such as molecular function, cellular process and biological process (Ness et al., 2011; Shi et al., 2011; Wang et al., 2010). WEGO (http://www.wego. genomics.org.cn) tool was used to perform the GO functional classification for all of the unigenes and to understand the distribution of the gene functions of this species at the macro level. The KEGG database (http://www.genome.jp/kegg/pathway.html) was used to annotate the pathway of these unigenes.
1.6 SSR mining
We employed MIcroSAtellite (MISA) (http://pgrc.ipk- gatersleben.de/misa/) for microsatellite mining which gives various statistical outputs of transcripts with useful information.
1.7 Plant transcription factor
PlantTFcat: An Online Plant Transcription Factor and Transcriptional Regulator Categorization and Analysis Tool used for identifying plant transcription factor in sequences (http://plantgrn.noble.org/PlantTFcat/). Transcription factor encoding transcripts were identified by sequence comparison to known transcription factor gene families.
2 Result and Discussions
2.1 NGS QC Toolkit
Sequence was filtered with this tool by removing adaptors and other contaminated materials then quality of sequence also checked with this tool and finally high quality filter sequence file considered for de novo sequence assembly (Table 1).

 

Table 1 NGS QC Toolkit Result


2
.2 De novo Sequence Assembly
CLC GENOMICS WORKBENCH 7 considered for de novo sequence assembly with by default parameters like Mismatch Cost = 2, Insertion Cost = 3, Deletion Cost = 3, Length Fraction = 0.5, Similarity Fraction = 0.8, Word size = 21 and finally 7256 contigs generated with average value of 445 by this software and other details are shown in Table 2.

 

Table 2 Contig measurement


3 Functional annotation with BLASTX and blast2GO
3.1 BLASTX
BLASTX was performed to align the contigs against non-redundant sequences database using an E value threshold of 10-6. Out of 7256 transcript contigs, 1983 were having BLAST hits to known proteins with high significant similarity and 167 had no BLAST hits (Table 3). Out of total transcripts contigs, Table 4 and Figure 1 shows that species distribution in which 2515 sequences showed significant similarity with Medicago truncatula and least similarity was found with Solanum lycopersicum (11).

 

Table 3 Blast Result


 

Table 4 Blast Result of Species Distribution


 

Figure 1 Blast Result of Species Distribution


3.2 Enzyme Code (EC) Classification
Enzyme classified with total of 575 sequences which is further classified into six classes which are of Oxidoreductases (148), Transferases (149), Hydrolases (179), Lyases (34), Isomerases (38) and Ligases (27) which is shown in Figure 2.

 

Figure 2 Enzyme Code (EC) Classification


3.3 Gene Ontology (GO) Classification
To functionally categorize Trigonella foenum-graecum L. transcript contigs, Gene Ontology (GO) terms were assigned to each assembled transcript contigs. Out of 7256 transcript contigs, 10179 unigenes were grouped into GO functional categories (http://www.geneontology.org), which are distributed under the three main categories of Molecular Function (2792), Biological Process (4407) and Cellular Components (2980) (Figure 3). Figure 4 which is output of WEGO tool; it shows that, Within the Molecular Function category, genes encoding binding proteins and proteins related to catalytic activity were the most enriched. Proteins related to metabolic processes and cellular processes were enriched in the Biological Process category. With regard to the Cellular Components category, the cell and cell part were the most highly represented categories.

 

Figure 3 Gene Ontology Result


 

Figure 4 WEGO Tool Result


A total of 192 unigenes were annotated with 87 pathways in the KEGG database (http://www.genome. jp/kegg/pathway.html). Many transcripts include various pathways like metabolic pathways, plant-pathogen interaction pathways, fatty acid metabolism pathway and fatty acid biosynthesis.
4 SSR mining
Microsatellite markers (SSR markers) are some of the most successful molecular markers in the construction of a Trigonella foenum-graecum L. genetic map and in diversity analysis (Zhang et al). For identification of SSRs, all transcripts were searched with perl script MISA. We identified a total of 3107 SSRs in 2191 transcripts (Table 5). The mono-nucleotide SSRs represented the largest fraction of SSRs identified followed by tri-nucleotide and di-nucleotide SSRs. Although only a small fraction of tetra-, penta- and hexa-nucleotide SSRs were identified in transcripts, the number is quite significant.

 

Table 5 Statistics of SSRs identified in transcripts


5 Plant Transcription Factor
Further, transcription factor encoding transcripts were identified by sequence comparison to known transcription factor gene families. Result shows that transcription factor genes distributed with at least 45 families were identified (Figure 5). The overall distribution of transcription factor encoding transcripts among the various known protein families is very similar with that of other legumes as predicted earlier (Libault et al., 2009).

 

Figure 5 Plant Transcription Factor Result


6
Conclusion
This study is focus on Trigonella foenum-graecum L. species (SRR066197) from NCBI database for de novo Transcriptome analysis by RNA-seq using next-generation 454 sequencing. In this study, we performed de novo functional annotation of the Trigonella foenum-graecum L. transcriptome without considering any reference species with significant non-redundant set of 7256 transcripts. The detailed analyses of the data set has provided several important features of Trigonella foenum-graecum L. transcriptome such as GC content, conserved genes across legumes and other plant species, assignment of functional categories by GO terms and identification of SSRs by MISA tool. Trigonella foenum-graecum L. contains many useful components like; Polysaccharide galactomannan, saponins (diosgenin, yamogenin, gitogenin, tigogenin, neotigogens), mucilage, volatile oils, alkaloids (choline and trigonelline). It is noted that this study of Trigonella foenum-graecum L. will be useful for further functional genomics studies as it includes useful information of each transcript.
Acknowledgement
We are heartily thankful to Prof. (Dr.) P.V. Virparia, Director, GDCST, Sardar Patel University, Vallabh Vidyanagar, for providing us facilities for the research work.
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