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De novo transcriptome assembly of the green alga Ankistrodesmus falcatus.

ABSTRACT: Ankistrodesmus falcatus is a globally distributed freshwater chlorophyte that is a candidate for biofuel production, is used to study the effects of toxins on aquatic communities, and is used as food in zooplankton research. Each of these research fields is transitioning to genomic tools. We created a reference transcriptome for of A. falcatus using NextGen sequencing and de novo assembly methods including Trinity, Velvet-Oases, and EvidentialGene. The assembled transcriptome has a total of 17,997 contigs, an N50 value of 2,462, and a GC content of 64.8%. BUSCO analysis recovered 83.3% of total chlorophyte BUSCOs and 82.5% of the eukaryotic BUSCOs. A portion (7.9%) of these supposedly single-copy genes were found to have transcriptionally active, distinct duplicates. We annotated the assembly using the dammit annotation pipeline, resulting in putative functional annotation for 68.89% of the assembly. Using available rbcL sequences from 16 strains (10 species) of Ankistrodesmus, we constructed a neighbor-joining phylogeny to illustrate genetic distances of our A. falcatus strain to other members of the genus. This assembly will be valuable for researchers seeking to identify Ankistrodesmus sequences in metatranscriptomic and metagenomic field studies and in experiments where separating expression responses of zooplankton and their algal food sources through bioinformatics is important.

SUBMITTER: Schomaker RA

PROVIDER: S-EPMC8121315 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MotivationThere are very few methods for de novo genome assembly based on the overlap graph approach. It is considered as giving more exact results than the so-called de Bruijn graph approach but in much greater time and of much higher memory usage. It is not uncommon that assembly methods involving the overlap graph model are not able to successfully compute greater data sets, mainly due to memory limitation of a computer. This was the reason for developing in last decades mainly de Bruijn-based assembly methods, fast and fairly accurate. However, the latter methods can fail for longer or more repetitive genomes, as they decompose reads to shorter fragments and lose a part of information. An efficient assembler for processing big data sets and using the overlap graph model is still looked out.ResultsWe propose a new genome-scale de novo assembler based on the overlap graph approach, designed for short-read sequencing data. The method, ALGA, incorporates several new ideas resulting in more exact contigs produced in short time. Among these ideas we have creation of a sparse but quite informative graph, reduction of the graph including a procedure referring to the problem of minimum spanning tree of a local subgraph, and graph traversal connected with simultaneous analysis of contigs stored so far. What is rare in genome assembly, the algorithm is almost parameter-free, with only one optional parameter to be set by a user. ALGA was compared with nine state-of-the-art assemblers in tests on genome-scale sequencing data obtained from real experiments on six organisms, differing in size, coverage, GC content, and repetition rate. ALGA produced best results in the sense of overall quality of genome reconstruction, understood as a good balance between genome coverage, accuracy, and length of resulting sequences. The algorithm is one of tools involved in processing data in currently realized national project Genomic Map of Poland.AvailabilityALGA is available at http://alga.put.poznan.pl.Supplementary informationSupplementary material is available at Bioinformatics online.

Project description:BACKGROUND: Neutral lipid storage is enhanced by nitrogen deprivation (ND) in numbers of green microalgal species. However, little is known about the metabolic pathways whose transcription levels are most significantly altered following ND in green microalgae, especially the nonmodel species. RESULTS: To start gaining knowledge on this, we performed transcriptome profiling of the nonmodel green microalga Botryosphaerella sudeticus cells in response to ND. Transcriptome of B. sudeticus is de novo assembled based on millions of HiSEQ short sequence reads using CLC Genomics Workbench software. The resulting non-redundant ESTs are annotated based on the best hits generated from the BLASTX homology comparison against the "best" proteins in the model microalgae Chlamydomonas reinhardtii and Chlorella variabilis. By using a pathway-based approach according to KEGG databases, we show that ESTs encoding ribosomal proteins and photosynthetic functions are the most abundantly expressed ESTs in the rapidly growing B. sudeticus cells. We find that ESTs encoding photosynthetic function but not the ribosomal proteins are most drastically downregulated upon ND. Notably, ESTs encoding lipid metabolic pathways are not significantly upregulated. Further analyses indicate that chlorophyll content is markedly decreased by 3-fold and total lipid content is only slightly increased by 50%, consistent with the transcriptional profiling. On the other hand, carbon content and photosynthesis efficiency are only marginally decreased by 7% and 20%, respectively, indicating that photosynthesis is only slightly reduced upon drastic downregulation of photosynthetic ESTs and chlorophyll content upon ND. In addition, TAG content is found to be greatly increased by 50-fold, though total lipid content is only slightly increased by 1.5-fold. CONCLUSIONS: Taken together, our results suggest that light-harvesting proteins and chlorophylls are in excess in B. sudeticus. Degradation of excess photosynthesis proteins is most likely a mechanism for recycling of nitrogen-rich molecules to synthesize new proteins for preparation of gametogenesis and zygospore formation in adaptation and survival upon ND. Furthermore, our analyses indicate that TAG accumulation is largely attributed to the modification of other pre-existing lipid molecules, rather than de novo synthesis. We propose that this is likely an evolutionarily conserved mechanism in many green microalgae species.

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De novo transcriptome assembly of the green alga Ankistrodesmus falcatus.

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