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| 2. |
- Westrin, Karl Johan, 1989-, et al.
(författare)
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ClusTrast : a short read de novo transcript isoform assembler guided by clustered contigs
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Transcriptome assembly from RNA-sequencing data in species without a reliable referencegenome has to be performed de novo, but studies have shown that de novo methods often have inadequateability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whosemain purpose is to produce a comprehensive set of transcript isoforms.Results: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq dataas input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guidingcontigs, assembles each clustered set of short reads individually, and merges the primary and clusterwiseassemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, andshowed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novoassemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end ofexpression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets.Reference transcripts were often (35–69% for the six datasets) reconstructed to at least 95% of their length byClusTrast, and more than half of reference transcripts (58–81%) were reconstructed with contigs that exhibitedpolymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using aunion of assembled transcripts from more than one assembly tool as primary assembly.Conclusion: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliablereference genome, in particular when the goal is to produce a comprehensive transcriptome set withpolymorphic variants.
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| 3. |
- Akhter, Shirin, et al.
(författare)
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Cone-setting in spruce is regulated by conserved elements of the age-dependent flowering pathway
- 2022
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Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 236:5, s. 1951-1963
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Tidskriftsartikel (refereegranskat)abstract
- Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.
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| 4. |
- Akhter, Shirin, et al.
(författare)
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Integrative Analysis of Three RNA Sequencing Methods Identifies Mutually Exclusive Exons of MADS-Box Isoforms During Early Bud Development in Picea abies
- 2018
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Ingår i: Frontiers in Plant Science. - : Frontiers Media S.A.. - 1664-462X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister Glade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this subclade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.
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| 5. |
- Akhter, Shirin, et al.
(författare)
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Transcriptome studies of the early cone-setting acrocona mutant provide evidence for a functional conservation of the age-dependent flowering pathway between angiosperms and gymnosperms.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- All seed plants go through a juvenile period before they initiate seed- and pollen-bearing organs and reproduce. Reproductive phase-change is well characterized in angiosperm model species, but much less well described in gymnosperms. Here, we utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies; a representative of the gymnosperm lineage. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. By sequence analysis of mRNA and microRNA transcripts, we demonstrate that orthologous components of the Age-dependent flowering pathway are active at the time of cone initiation. We show that a member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL7, is active in reproductive meristems, whereas a putative negative regulator of PaSPL7, microRNA156 is upregulated in vegetative meristem. By allele-specific assembly, we also identify a short nucleotide polymorphism (SNP) in the miRNA156 binding of PaSPL7. By genotyping a segregating population of inbred acrocona trees, we show a clear co-segregation between the early cone-setting phenotype and trees homozygous for the SNP. Hence, the data presented demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating cone-setting in the conifer P. abies.
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| 6. |
- Westrin, Karl Johan, 1989-, et al.
(författare)
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ClusTrast : a short read de novo transcript isoform assembler guided by clustered contigs
- 2024
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Ingår i: BMC Bioinformatics. - : Springer Nature. - 1471-2105. ; 25:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Transcriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate ability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whose main purpose is to produce a comprehensive set of transcript isoforms. Results: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq data as input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guiding contigs, assembles each clustered set of short reads individually, and merges the primary and clusterwise assemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end of expression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets. Reference transcripts were often (35–69% for the six datasets) reconstructed to at least 95% of their length by ClusTrast, and more than half of reference transcripts (58–81%) were reconstructed with contigs that exhibited polymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using a union of assembled transcripts from more than one assembly tool as primary assembly. Conclusion: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliable reference genome, in particular when the goal is to produce a comprehensive transcriptome set with polymorphic variants.
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| 7. |
- Westrin, Karl Johan, 1989-, et al.
(författare)
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Short read de novo transcript isoform assembly guided by clustered contigs
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Motivation: Transcriptome assembly in species without a reference genome has to beperformed de novo, which requires a deeper sequencing than a reference based approach would require. In turn, this makes the study of alternative splicing in such species difficult,particularly for lowly expressed isoforms. Sequencing of full-length transcripts using longreads could improve this, but such techniques are still either expensive or error-prone.Result: In this paper we present the novel de novo transcript isoform assembler ClusTrast, which clusters a set of guiding contigs – e.g. long reads or a previous assembly – into(supposed) gene families. ClusTrast will then align the short reads to the guiding contigs,and thus the short reads are clustered. Finally, ClusTrast will assemble each cluster ofshort reads individually. Tested on real datasets from human, mouse, arabidopsis, rice, zebrafish and Norway spruce, ClusTrast managed to recover more of the known expressed isoforms than any of the other tested de novo assemblers, while mostly retaining precision. Therefore, we presume that ClusTrast can be a useful tool for studying alternative splicing.Contact: olofem@kth.se
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