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Comprehensive Stress-Based De Novo Transcriptome Assembly and Annotation of Guar (Cyamopsis tetragonoloba (L.) Taub.): An Important Industrial and Forage Crop.

Fahad Al-Qurainy | Aref Alshameri | Abdel-Rhman Gaafar | Salim Khan | Mohammad Nadeem | Abdulhafed Abdullah Alameri | Mohamed Tarroum | Muhammad Ashraf
International journal of genomics | 2019

The forage crop Guar (Cyamopsis tetragonoloba (L.) Taub.) has the ability to endure heat, drought, and mild salinity. A complete image on its genic architecture will promote our understanding about gene expression networks and different tolerance mechanisms at the molecular level. Therefore, whole mRNA sequence approach on the Guar plant was conducted to provide a snapshot of the mRNA information in the cell under salinity, heat, and drought stresses to be integrated with previous transcriptomic studies. RNA-Seq technology was employed to perform a 2 × 100 paired-end sequencing using an Illumina HiSeq 2500 platform for the transcriptome of leaves of C. tetragonoloba under normal, heat, drought, and salinity conditions. Trinity was used to achieve a de novo assembly followed by gene annotation, functional classification, metabolic pathway analysis, and identification of SSR markers. A total of 218.2 million paired-end raw reads (~44 Gbp) were generated. Of those, 193.5M paired-end reads of high quality were used to reconstruct a total of 161,058 transcripts (~266 Mbp) with N50 of 2552 bp and 61,508 putative genes. There were 6463 proteins having >90% full-length coverage against the Swiss-Prot database and 94% complete orthologs against Embryophyta. Approximately, 62.87% of transcripts were blasted, 50.46% mapped, and 43.50% annotated. A total of 4715 InterProScan families, 3441 domains, 74 repeats, and 490 sites were detected. Biological processes, molecular functions, and cellular components comprised 64.12%, 25.42%, and 10.4%, respectively. The transcriptome was associated with 985 enzymes and 156 KEGG pathways. A total of 27,066 SSRs were gained with an average frequency of one SSR/9.825 kb in the assembled transcripts. This resulting data will be helpful for the advanced analysis of Guar to multi-stress tolerance.

Pubmed ID: 31687376

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This is a list of tools and resources that we have found mentioned in this publication.


BLASTN (tool)

RRID:SCR_001598

Web application to search nucleotide databases using a nucleotide query. Algorithms: blastn, megablast, discontiguous megablast.

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BLASTX (tool)

RRID:SCR_001653

Web application to search protein databases using a translated nucleotide query. Translated BLAST services are useful when trying to find homologous proteins to a nucleotide coding region. Blastx compares translational products of the nucleotide query sequence to a protein database. Because blastx translates the query sequence in all six reading frames and provides combined significance statistics for hits to different frames, it is particularly useful when the reading frame of the query sequence is unknown or it contains errors that may lead to frame shifts or other coding errors. Thus blastx is often the first analysis performed with a newly determined nucleotide sequence and is used extensively in analyzing EST sequences. This search is more sensitive than nucleotide blast since the comparison is performed at the protein level.

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Gene Ontology (tool)

RRID:SCR_002811

Computable knowledge regarding functions of genes and gene products. GO resources include biomedical ontologies that cover molecular domains of all life forms as well as extensive compilations of gene product annotations to these ontologies that provide largely species-neutral, comprehensive statements about what gene products do. Used to standardize representation of gene and gene product attributes across species and databases.

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PRINSEQ (tool)

RRID:SCR_005454

A publicly available tool that is able to filter, reformat and trim your genomic and metagenomic sequence data and provide you summary statistics for your sequence data. The interactive web interface facilitates visualizations of the results and export functionality for subsequent data processing. The standalone lite version is written in Perl and does not require any non-core Perl modules. The lite version is primarily designed for data preprocessing and does not generate summary statistics in graphical form.

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Bowtie (tool)

RRID:SCR_005476

Software ultrafast memory efficient tool for aligning sequencing reads. Bowtie is short read aligner.

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Blast2GO (tool)

RRID:SCR_005828

An ALL in ONE tool for functional annotation of (novel) sequences and the analysis of annotation data. Blast2GO (B2G) joins in one universal application similarity search based GO annotation and functional analysis. B2G offers the possibility of direct statistical analysis on gene function information and visualization of relevant functional features on a highlighted GO direct acyclic graph (DAG). Furthermore B2G includes various statistics charts summarizing the results obtained at BLASTing, GO-mapping, annotation and enrichment analysis (Fisher''''s Exact Test). All analysis process steps are configurable and data import and export are supported at any stage. The application also accepts pre-existing BLAST or annotation files and takes them to subsequent steps. The tool offers a very suitable platform for high throughput functional genomics research in non-model species. B2G is a species-independent, intuitive and interactive desktop application which allows monitoring and comprehending the whole annotation and analysis process supported by additional features like GO Slim integration, evidence code (EC) consideration, a Batch-Mode or GO-Multilevel-Pies. Platform: Windows compatible, Mac OS X compatible, Linux compatible, Unix compatible

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InterPro (tool)

RRID:SCR_006695

Service providing functional analysis of proteins by classifying them into families and predicting domains and important sites. They combine protein signatures from a number of member databases into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool. This integrated database of predictive protein signatures is used for the classification and automatic annotation of proteins and genomes. InterPro classifies sequences at superfamily, family and subfamily levels, predicting the occurrence of functional domains, repeats and important sites. InterPro adds in-depth annotation, including GO terms, to the protein signatures. You can access the data programmatically, via Web Services. The member databases use a number of approaches: # ProDom: provider of sequence-clusters built from UniProtKB using PSI-BLAST. # PROSITE patterns: provider of simple regular expressions. # PROSITE and HAMAP profiles: provide sequence matrices. # PRINTS provider of fingerprints, which are groups of aligned, un-weighted Position Specific Sequence Matrices (PSSMs). # PANTHER, PIRSF, Pfam, SMART, TIGRFAMs, Gene3D and SUPERFAMILY: are providers of hidden Markov models (HMMs). Your contributions are welcome. You are encouraged to use the ''''Add your annotation'''' button on InterPro entry pages to suggest updated or improved annotation for individual InterPro entries.

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CD-HIT (tool)

RRID:SCR_007105

THIS RESOURCE IS NO LONGER IN SERVICE. Documented on February 28,2023. Software program for clustering biological sequences with many applications in various fields such as making non-redundant databases, finding duplicates, identifying protein families, filtering sequence errors and improving sequence assembly etc. It is very fast and can handle extremely large databases. CD-HIT helps to significantly reduce the computational and manual efforts in many sequence analysis tasks and aids in understanding the data structure and correct the bias within a dataset. The CD-HIT package has CD-HIT, CD-HIT-2D, CD-HIT-EST, CD-HIT-EST-2D, CD-HIT-454, CD-HIT-PARA, PSI-CD-HIT, CD-HIT-OTU and over a dozen scripts. * CD-HIT (CD-HIT-EST) clusters similar proteins (DNAs) into clusters that meet a user-defined similarity threshold. * CD-HIT-2D (CD-HIT-EST-2D) compares 2 datasets and identifies the sequences in db2 that are similar to db1 above a threshold. * CD-HIT-454 identifies natural and artificial duplicates from pyrosequencing reads. * CD-HIT-OTU cluster rRNA tags into OTUs The usage of other programs and scripts can be found in CD-HIT user''s guide. CD-HIT was originally developed by Dr. Weizhong Li at Dr. Adam Godzik''s Lab at the Burnham Institute (now Sanford-Burnham Medical Research Institute).

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QIAGEN (tool)

RRID:SCR_008539

A commercial organization which provides assay technologies to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.

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MISA (tool)

RRID:SCR_010765

Software tool that allows the identification and localization of perfect microsatellites as well as compound microsatellites which are interrupted by a certain number of bases.

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OrthoDB (tool)

RRID:SCR_011980

Database of orthologous protein coding genes across vertebrates, arthropods, fungi, basal metazoans, and bacteria.

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Trinity (tool)

RRID:SCR_013048

Software for the efficient and robust de novo reconstruction of transcriptomes from RNA-seq data.

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Macrogen (tool)

RRID:SCR_014454

A company that provides a variety of next generation sequencing services. The company provides researchers with whole genome resequencing, exome sequencing, targeted sequencing, transcriptomics, and epigenome sequencing.

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FastQC (tool)

RRID:SCR_014583

Quality control software that perform checks on raw sequence data coming from high throughput sequencing pipelines. This software also provides a modular set of analyses which can give a quick impression of the quality of the data prior to further analysis.

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BUSCO (tool)

RRID:SCR_015008

Software tool to quantitatively measure genome assembly and annotation completeness based on evolutionarily informed expectations of gene content.

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