scholarly journals Raw transcriptomics data to gene specific SSRs: a validated free bioinformatics workflow for biologists

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
D. N. U. Naranpanawa ◽  
C. H. W. M. R. B. Chandrasekara ◽  
P. C. G. Bandaranayake ◽  
A. U. Bandaranayake
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
Transcriptome Assembly ◽  
Low Cost ◽  
Santalum Album ◽  
Sequencing Data ◽  
Illumina Hiseq ◽  
Tissue Samples ◽  
Downstream Analysis ◽  
Bioinformatics Workflow

Abstract Recent advances in next-generation sequencing technologies have paved the path for a considerable amount of sequencing data at a relatively low cost. This has revolutionized the genomics and transcriptomics studies. However, different challenges are now created in handling such data with available bioinformatics platforms both in assembly and downstream analysis performed in order to infer correct biological meaning. Though there are a handful of commercial software and tools for some of the procedures, cost of such tools has made them prohibitive for most research laboratories. While individual open-source or free software tools are available for most of the bioinformatics applications, those components usually operate standalone and are not combined for a user-friendly workflow. Therefore, beginners in bioinformatics might find analysis procedures starting from raw sequence data too complicated and time-consuming with the associated learning-curve. Here, we outline a procedure for de novo transcriptome assembly and Simple Sequence Repeats (SSR) primer design solely based on tools that are available online for free use. For validation of the developed workflow, we used Illumina HiSeq reads of different tissue samples of Santalum album (sandalwood), generated from a previous transcriptomics project. A portion of the designed primers were tested in the lab with relevant samples and all of them successfully amplified the targeted regions. The presented bioinformatics workflow can accurately assemble quality transcriptomes and develop gene specific SSRs. Beginner biologists and researchers in bioinformatics can easily utilize this workflow for research purposes.

scholarly journals De novo transcriptome assembly of Premnotrypes vorax (Coleoptera: Curculionidae)

2020 ◽  
Author(s):  
Luisa-Fernanda Velásquez C. ◽  
Pablo Emiliano Canton ◽  
Alejandro Sánchez-Flores ◽  
Alejandra Bravo ◽  
Jairo Cerón
Keyword(s):  
Biological Control ◽  
Control Strategy ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Insect Pest ◽  
Illumina Hiseq ◽  
Tissue Samples ◽  
Cry Toxin ◽  
Illumina Hiseq Platform ◽  
Potato Crops

Abstract Objective: Premnotrypes vorax (P. vorax) is an insect pest that causes significant losses to potato crops in Colombia. Currently, the insect control is mainly done by using highly toxic chemical insecticides and there are no reports of any commercial biological control strategy against this pest. Hence, the objective of this study was to characterize the insect genetic expression to search for genes that could codify for Bacillus thuringiensis Cry toxin receptors. Using an RNA-seq approach, we sequenced the mRNA from the insect tissue, performed a de novo assembly and analyzed the reconstructed transcriptome of P. vorax. To our knowledge, this is the first genetic report of this endemic insect which will set the basis of a possible biological control strategy.Results: The transcriptome data was obtained from dissected midgut tissue samples of P. vorax larvae. The isolated RNA was isolated and sequenced using the Illumina HiSeq platform with a configuration of 2x150pb reads. A total of 383,552,246 reads were obtained and subsequently a quality and cleaning process was performed through FastQC and Trimmomatic software, respectively. A novo assembly was done using the Trinity software, obtaining a transcriptome assembly with 25,631 genes that showed at least one annotation record, resulting in 74,984 transcript isoforms.

scholarly journals Enhancing De Novo Transcriptome Assembly by Incorporating Multiple Overlap Sizes

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Chien-Chih Chen ◽  
Wen-Dar Lin ◽  
Yu-Jung Chang ◽  
Chuen-Liang Chen ◽  
Jan-Ming Ho
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
Transcriptome Assembly ◽  
Next Generation ◽  
Sequencing Data ◽  
Evaluation Tool ◽  
Coverage Depth ◽  
Sequencing Platform ◽  
Assembly Algorithms ◽  
The Relationship

Background. The emergence of next-generation sequencing platform gives rise to a new generation of assembly algorithms. Compared with the Sanger sequencing data, the next-generation sequence data present shorter reads, higher coverage depth, and different error profiles. These features bring new challenging issues for de novo transcriptome assembly. Methodology. To explore the influence of these features on assembly algorithms, we studied the relationship between read overlap size, coverage depth, and error rate using simulated data. According to the relationship, we propose a de novo transcriptome assembly procedure, called Euler-mix, and demonstrate its performance on a real transcriptome dataset of mice. The simulation tool and evaluation tool are freely available as open source. Significance. Euler-mix is a straightforward pipeline; it focuses on dealing with the variation of coverage depth of short reads dataset. The experiment result showed that Euler-mix improves the performance of de novo transcriptome assembly.

scholarly journals TrancriptomeReconstructoR, A Data-Driven Annotation of Complex Transcriptomes

2020 ◽  
Author(s):  
Maxim Ivanov ◽  
Albin Sandelin ◽  
Sebastian Marquardt
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
R Package ◽  
Sequence Information ◽  
Rna Seq ◽  
Sequencing Data ◽  
Gene Model ◽  
Preparation Methods ◽  
Downstream Analysis

Abstract Background: The quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data. Results: We developed TranscriptomeReconstructoR, an R package which implements a pipeline for automated transcriptome annotation. It relies on integrating features from independent and complementary datasets: i) full-length RNA-seq for detection of splicing patterns and ii) high-throughput 5' and 3' tag sequencing data for accurate definition of gene borders. The pipeline can also take a nascent RNA-seq dataset to supplement the called gene model with transient transcripts.We reconstructed de novo the transcriptional landscape of wild type Arabidopsis thaliana seedlings as a proof-of-principle. A comparison to the existing transcriptome annotations revealed that our gene model is more accurate and comprehensive than the two most commonly used community gene models, TAIR10 and Araport11. In particular, we identify thousands of transient transcripts missing from the existing annotations. Our new annotation promises to improve the quality of A.thaliana genome research.Conclusions: Our proof-of-concept data suggest a cost-efficient strategy for rapid and accurate annotation of complex eukaryotic transcriptomes. We combine the choice of library preparation methods and sequencing platforms with the dedicated computational pipeline implemented in the TranscriptomeReconstructoR package. The pipeline only requires prior knowledge on the reference genomic DNA sequence, but not the transcriptome. The package seamlessly integrates with Bioconductor packages for downstream analysis.

EdClust: A heuristic sequence clustering method with higher sensitivity

2021 ◽  
Author(s):  
Ming Cao ◽  
Qinke Peng ◽  
Ze-Gang Wei ◽  
Fei Liu ◽  
Yi-Fan Hou
Keyword(s):  
Large Scale ◽  
Sequence Data ◽  
Clustering Algorithms ◽  
Clustering Methods ◽  
Sequencing Data ◽  
Clustering Method ◽  
Cluster Number ◽  
Sequence Clustering ◽  
Downstream Analysis ◽  
Heuristic Clustering

The development of high-throughput technologies has produced increasing amounts of sequence data and an increasing need for efficient clustering algorithms that can process massive volumes of sequencing data for downstream analysis. Heuristic clustering methods are widely applied for sequence clustering because of their low computational complexity. Although numerous heuristic clustering methods have been developed, they suffer from two limitations: overestimation of inferred clusters and low clustering sensitivity. To address these issues, we present a new sequence clustering method (edClust) based on Edlib, a C/C[Formula: see text] library for fast, exact semi-global sequence alignment to group similar sequences. The new method edClust was tested on three large-scale sequence databases, and we compared edClust to several classic heuristic clustering methods, such as UCLUST, CD-HIT, and VSEARCH. Evaluations based on the metrics of cluster number and seed sensitivity (SS) demonstrate that edClust can produce fewer clusters than other methods and that its SS is higher than that of other methods. The source codes of edClust are available from https://github.com/zhang134/EdClust.git under the GNU GPL license.

De novo transcriptome assembly and analysis of the codon usage bias of the MADS-box gene family in Cymbidium kanran

2019 ◽  
Vol 79 (02) ◽  
Author(s):  
Boyun Yang ◽  
Huolin Luo ◽  
Yuan Tao ◽  
Wenjing Yu ◽  
Liping Luo
Keyword(s):  
Codon Usage ◽  
Codon Usage Bias ◽  
De Novo ◽  
Average Length ◽  
Transcriptome Assembly ◽  
Mads Box ◽  
Illumina Hiseq ◽  
Optimal Codons ◽  
Mads Box Gene ◽  
New Varieties

Cymbidium kanran is an important commercially grown member of the Chinese orchid family. However, little information regarding the molecular biology of this species is available. In this study, the C. kanran root, shoot, stem, leaf, and flower transcriptomes were sequenced with the Illumina HiSeq 4000 system, which resulted in 8.9 Gb of clean reads that were assembled into 74,620 unigenes, with an average length and N50 of 983 bp and 1,640 bp, respectively. The screening of seven databases (NR, NT, GO, KOG, KEGG, Swiss-Prot, and InterPro) for similar sequences resulted in the functional annotation of 49,813 unigenes. Additionally, 173 MADS-box genes, which help to control major aspects of plant development, were identified and their codon usage bias was analyzed. Only 26 genes had a low ENC (less than or equal to 35), suggesting the codon usage bias was weak. Base mutations were the major determinants of codon usage, although natural selection pressure also influenced codon usage bias. Moreover, 22 optimal codons were identified based on ΔRSCU, and 20 codons ended with A/U. The results of this study provide the foundation for the molecular breeding of new varieties

Computational Approaches for Transcriptome Assembly Based on Sequencing Technologies

2020 ◽  
Vol 15 (1) ◽  
pp. 2-16
Author(s):  
Yuwen Luo ◽  
Xingyu Liao ◽  
Fang-Xiang Wu ◽  
Jianxin Wang
Keyword(s):  
De Novo ◽  
Transcriptome Assembly ◽  
Critical Role ◽  
High Sensitivity ◽  
Biological Properties ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Massive Sequencing ◽  
Generation Sequencing

Transcriptome assembly plays a critical role in studying biological properties and examining the expression levels of genomes in specific cells. It is also the basis of many downstream analyses. With the increase of speed and the decrease in cost, massive sequencing data continues to accumulate. A large number of assembly strategies based on different computational methods and experiments have been developed. How to efficiently perform transcriptome assembly with high sensitivity and accuracy becomes a key issue. In this work, the issues with transcriptome assembly are explored based on different sequencing technologies. Specifically, transcriptome assemblies with next-generation sequencing reads are divided into reference-based assemblies and de novo assemblies. The examples of different species are used to illustrate that long reads produced by the third-generation sequencing technologies can cover fulllength transcripts without assemblies. In addition, different transcriptome assemblies using the Hybrid-seq methods and other tools are also summarized. Finally, we discuss the future directions of transcriptome assemblies.

scholarly journals NanoGalaxy: Nanopore long-read sequencing data analysis in Galaxy

GigaScience ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Willem de Koning ◽  
Milad Miladi ◽  
Saskia Hiltemann ◽  
Astrid Heikema ◽  
John P Hays ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Bioinformatics Analysis ◽  
De Novo ◽  
Sequence Data ◽  
Ease Of Use ◽  
Easy Access ◽  
Complex Data ◽  
Sequencing Data ◽  
Long Read ◽  
Sequencing Platforms

Abstract Background Long-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers. Results The Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads. Conclusions A range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

scholarly journals Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae

2018 ◽  
Vol 5 (12) ◽  
pp. 181247 ◽  
Author(s):  
Tengfei Liu ◽  
Ziyao Liu ◽  
Xueyan Yao ◽  
Ying Huang ◽  
Qingsong Qu ◽  
...  
Keyword(s):  
Fruiting Body ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Quantitative Polymerase Chain Reaction ◽  
Nucleotide Metabolism ◽  
Differentially Expressed ◽  
Parasitic Fungus ◽  
Active Constituent ◽  
Illumina Hiseq ◽  
Cordyceps Cicadae

Cordyceps cicadae (Chanhua) is a parasitic fungus that grows on Cicada flammata larvae and is used to relieve exhaustion and treat numerous diseases, in part through its active constituent, cordycepin. We used de novo Illumina HiSeq 4000 sequencing to obtain transcriptomes of C. cicadae mycelium, fruiting body, and sclerotium, and identify differentially expressed genes. In the mycelium versus sclerotium libraries, 1576 upregulated and 2300 downregulated genes were identified. In the mycelium versus fruiting body and fruiting body versus sclerotium body libraries, 1604 and 1474 upregulated and 1365 and 1320 downregulated genes, respectively, were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified 19 genes differentially expressed in mycelium versus fruiting body as related to the purine pathway, along with 28 and 16 genes differentially expressed in the mycelium versus sclerotium and fruiting body versus sclerotium groups, respectively. Gene expression of six key enzymes was validated by quantitative polymerase chain reaction. Specifically, 5′-nucleotidase (c62060g1) and adenosine deaminase (c35629g1) in purine nucleotide metabolism, which are involved in cordycepin biosynthesis, were significantly upregulated in the sclerotium group. These findings improved our understanding of genes involved in the biosynthesis of cordycepin and other characteristic secondary metabolites in C. cicadae .

scholarly journals Isolation and characterization of polymorphic microsatellite markers in Toxicodendron vernicifluum

2018 ◽  
Vol 54 (No. 1) ◽  
pp. 17-25 ◽  
Author(s):  
D.-D. Vu ◽  
T.T.-X. Bui ◽  
T.H.-N. Nguyen ◽  
S.N.M. Shah ◽  
N.-H. Vu ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Expressed Sequence Tag ◽  
De Novo ◽  
Average Length ◽  
Polymorphic Information Content ◽  
Shaanxi Province ◽  
Illumina Hiseq ◽  
Tissue Samples ◽  
Isolation And Characterization ◽  
Toxicodendron Vernicifluum

A total 20 074 230 sequencing reads were generated by Illumina HiSeq<sup>™ </sup>2500 from three different Toxicodendron vernicifluum tissue samples. In total, 48 693 unigenes with an average length of 703.34 bp were obtained by de novo assembly. 3392 potential EST-SSRs (expressed sequence tag-simple sequence repeat) were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 80 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 14 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism within the lacquer tree population in Langao, Shaanxi province, China. There were high genetic diversities (number of alleles per locus (A) = 2.93, polymorphic information content (PIC) = 0.53, observed heterozygosity (Ho) = 0.62 and expected heterozygosity (He) = 0.85) in the lacquer tree natural population. The four loci were significantly deviated from Hardy-Weinberg equilibrium. These results suggested high homozygosity in the population and low or deficiency in heterozygosity (inbreeding coefficient (Fis) = 0.27). These polymorphic EST-SSR markers will provide the base for further studies of genetic structure and breeding in T. vernicifluum.

scholarly journals Aquila_stLFR: assembly based variant calling package for stLFR and hybrid assembly for linked-reads

2019 ◽  
Author(s):  
Xin Zhou ◽  
Lu Zhang ◽  
Xiaodong Fang ◽  
Yichen Liu ◽  
David L. Dill ◽  
...  
Keyword(s):  
De Novo ◽  
Low Cost ◽  
Variant Calling ◽  
Hybrid Assembly ◽  
Structural Variants ◽  
Sequencing Data ◽  
Single Tube ◽  
Large Numbers ◽  
Key Characteristics ◽  
Hybrid Assemblies

AbstractHuman diploid genome assembly enables identifying maternal and paternal genetic variations. Algorithms based on 10x linked-read sequencing have been developed for de novo assembly, variant calling and haplotyping. Another linked-read technology, single tube long fragment read (stLFR), has recently provided a low-cost single tube solution that can enable long fragment data. However, no existing software is available for human diploid assembly and variant calls. We develop Aquila stLFR to adapt to the key characteristics of stLFR. Aquila stLFR assembles near perfect diploid assembled contigs, and the assembly-based variant calling shows that Aquila stLFR detects large numbers of structural variants which were not easily spanned by Illumina short-reads. Furthermore, the hybrid assembly mode Aquila hybrid allows a hybrid assembly based on both stLFR and 10x linked-reads libraries, demonstrating that these two technologies can always be complementary to each other for assembly to improve contiguity and the variants detection, regardless of assembly quality of the library itself from single sequencing technology. The overlapped structural variants (SVs) from two independent sequencing data of the same individual, and the SVs from hybrid assemblies provide us a high-confidence profile to study them.AvailabilitySource code and documentation are available on https://github.com/maiziex/Aquila_stLFR.

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