EBARDenovo: highly accurate <i>de novo</i> assembly of RNA-Seq with efficient chimera-detection

Chu, Hsueh-Ting; Hsiao, William W. L.; Chen, Jen-Chih; Yeh, Tze-Jung; Tsai, Mong-Hsun; Lin, Han; Liu, Yen-Wenn; Lee, Sheng-An; Chen, Chaur-Chin; Tsao, Theresa Tsun-Hui; Kao, Cheng−Yan

doi:10.1093/bioinformatics/btt092

Cited by 32 publications

(19 citation statements)

References 23 publications

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“…Highly abundant transcripts may have some error-containing reads that recur with sufficient frequency that they are assembled into distinct contigs, and gene families with many members could generate chimeric contigs if they have identical subsequences that are longer than the k -mers used for assembly. In addition, templates derived from more than one gene may arise during the reverse-transcription or PCR steps of library preparation; the resulting fusion reads may lead to incorrect assembly of contigs, as well as increase the proportion of such contigs substantially when the Oases merge function is used [from 3.6% to 12.2% in one reported case (Chu et al 2013)]. The question about accession numbers can perhaps be explained by similar mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

Lehnert

Mouchka

Burriesci

et al. 2014

G3 Genes|Genomes|Genetics

171

248

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Coral reefs provide habitats for a disproportionate number of marine species relative to the small area of the oceans that they occupy. The mutualism between the cnidarian animal hosts and their intracellular dinoflagellate symbionts provides the nutritional foundation for coral growth and formation of reef structures, because algal photosynthesis can provide >90% of the total energy of the host. Disruption of this symbiosis (“coral bleaching”) is occurring on a large scale due primarily to anthropogenic factors and poses a major threat to the future of coral reefs. Despite the importance of this symbiosis, the cellular mechanisms involved in its establishment, maintenance, and breakdown remain largely unknown. We report our continued development of genomic tools to study these mechanisms in Aiptasia, a small sea anemone with great promise as a model system for studies of cnidarian–dinoflagellate symbiosis. Specifically, we have generated de novo assemblies of the transcriptomes of both a clonal line of symbiotic anemones and their endogenous dinoflagellate symbionts. We then compared transcript abundances in animals with and without dinoflagellates. This analysis identified >900 differentially expressed genes and allowed us to generate testable hypotheses about the cellular functions affected by symbiosis establishment. The differentially regulated transcripts include >60 encoding proteins that may play roles in transporting various nutrients between the symbiotic partners; many more encoding proteins functioning in several metabolic pathways, providing clues regarding how the transported nutrients may be used by the partners; and several encoding proteins that may be involved in host recognition and tolerance of the dinoflagellate.

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Section: Discussionmentioning

confidence: 99%

Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

Lehnert

Mouchka

Burriesci

et al. 2014

G3 Genes|Genomes|Genetics

171

248

View full text Add to dashboard Cite

show abstract

“…Sequence repeats, homologous genes and artificial chimeric reads caused by PCR amplification result in many chimeric contigs (Chu et al 2013). It was difficult to judge the gene structure of these contigs, thus we omitted these contigs for the process of ORF detection.…”

Section: Discussionmentioning

confidence: 99%

De novo assembly of Zea nicaraguensis root transcriptome identified 5 261 full-length transcripts

Jiang

Liu

et al. 2016

Journal of Integrative Agriculture

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Zea nicaraguensis, a wild relative of cultivated maize (Zea mays subsp. mays), is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verification to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L -1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-redundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology (GO) annotation, 2 354 received cluster of orthologous groups (COG) terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes (KEGG) Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.

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“…However, any other assembly tool, such as EBARDenovo (16), Oases (17), or Trinity (18), could be used in this workflow. The first step of the qNovo4 assembly trimmed the reads based on a user-defined Phred score and ambiguous base (N) content.…”

Section: Methodsmentioning

confidence: 99%

A Workflow to Increase Verification Rate of Chromosomal Structural Rearrangements Using High-Throughput Next-Generation Sequencing

et al. 2014

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Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

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EBARDenovo: highly accurate de novo assembly of RNA-Seq with efficient chimera-detection

Abstract: Supplementary data are available at Bioinformatics online.

Cited by 32 publications

References 23 publications

Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

De novo assembly of Zea nicaraguensis root transcriptome identified 5 261 full-length transcripts

A Workflow to Increase Verification Rate of Chromosomal Structural Rearrangements Using High-Throughput Next-Generation Sequencing

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