scholarly journals Chromosome scale assembly of allopolyploid genome of the diatom Fistulifera solaris

2021 ◽  
Author(s):  
Yoshiaki Maeda ◽  
Kahori Watanabe ◽  
Ryosuke Kobayashi ◽  
Tomoko Yoshino ◽  
Chris Bowler ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Rational Design ◽  
Large Scale ◽  
Biofuel Production ◽  
Bacterial Conjugation ◽  
Nanopore Sequencing ◽  
Chromosomal Structure ◽  
Sequencing Platform ◽  
Artificial Chromosomes ◽  
Long Read

Microalgae including diatoms are of interest for environmentally-friendly manufacturing such as biofuel production. However, only a very few of their genomes have been elucidated owing to their diversified and complex evolutionary history. The genome of the marine oleaginous diatom Fistulifera solaris, an allopolyploid diatom possessing two subgenomes, has been analyzed previously by pyrosequencing. However, many unsolved regions and unconnected scaffolds remained. Here we report the entire chromosomal structure of the genome of F. solaris strain JPCC DA0580 using a long-read nanopore sequencing platform. From just one single run using a MinION flow-cell, the chromosome scale assembly with telomere-to-telomere resolution was achieved for 41 out of 44 chromosomes. Centromere regions were also predicted from the chromosomes, and we discovered conserved motifs in the predicted regions. The function of the motifs was experimentally confirmed by successful transformation of the diatom via bacterial conjugation. This discovery provides insights into chromosome replication, facilitating the rational design of artificial chromosomes for large-scale metabolic engineering of diatoms. The chromosome scale assembly also suggests the potential existence of multi-copy mini-chromosomes and tandemly repeated lipogenesis genes related to the oleaginous phenotype of F. solaris. The nanopore sequencing also solved the sequential arrangement of the repeat region in the F. solaris mitochondrial genome. Findings of this study will be useful to understand and further engineer the oleaginous phenotype of F. solaris.

scholarly journals The human ribosomal DNA array is composed of highly homogenized tandem clusters

2021 ◽  
Author(s):  
Yutaro Hori ◽  
Akira Shimamoto ◽  
Takehiko Kobayashi
Keyword(s):  
Ribosomal Dna ◽  
Copy Number ◽  
Large Scale ◽  
Human Cells ◽  
Nanopore Sequencing ◽  
Cellular Functions ◽  
Rdna Cluster ◽  
Repetitive Nature ◽  
Long Read ◽  
Rdna Copy

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

scholarly journals circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zelin Liu ◽  
Changyu Tao ◽  
Shiwei Li ◽  
Minghao Du ◽  
Yongtai Bai ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Full Length ◽  
Circular Rnas ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Functional Studies ◽  
Sequencing Method ◽  
Long Read ◽  
Fine Tune

Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

scholarly journals Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
V. Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Content Type ◽  
Sequencing Platform ◽  
Sequencing Studies ◽  
Long Read

ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.

scholarly journals circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

2021 ◽  
Author(s):  
Zelin Liu ◽  
Changyu Tao ◽  
Shiwei Li ◽  
Minghao Du ◽  
Yongtai Bai ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Full Length ◽  
Circular Rnas ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Functional Studies ◽  
Sequencing Method ◽  
Long Read ◽  
Fine Tune

Circular RNAs (circRNAs) act through multiple mechanisms with their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a great challenge, which hinders comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline circfull to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. Benefiting from rolling circles and long-read sequencing, circFL-seq showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to short-read RNA sequencing. The concordance of RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of cancer-related fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

The sugar and alcohol industry in the biofuels and cogeneration era: a paradigm change (part II)

2014 ◽  
pp. 97-104 ◽  
Author(s):  
Electo Eduardo Silv Lora ◽  
Mateus Henrique Rocha ◽  
José Carlos Escobar Palacio ◽  
Osvaldo José Venturini ◽  
Maria Luiza Grillo Renó ◽  
...  
Keyword(s):  
Large Scale ◽  
New Technologies ◽  
Process Integration ◽  
Animal Feed ◽  
Raw Materials ◽  
Biofuel Production ◽  
New Paradigm ◽  
Alcohol Industry ◽  
Feed Production ◽  
Steam Parameters

The aim of this paper is to discuss the major technological changes related to the implementation of large-scale cogeneration and biofuel production in the sugar and alcohol industry. The reduction of the process steam consumption, implementation of new alternatives in driving mills, the widespread practice of high steam parameters use in cogeneration facilities, the insertion of new technologies for biofuels production (hydrolysis and gasification), the energy conversion of sugarcane trash and vinasse, animal feed production, process integration and implementation of the biorefinery concept are considered. Another new paradigm consists in the wide spreading of sustainability studies of products and processes using the Life Cycle Assessment (LCA) and the implementation of sustainability indexes. Every approach to this issue has as an objective to increase the economic efficiency and the possibilities of the sugarcane as a main source of two basic raw materials: fibres and sugar. The paper briefly presents the concepts, indicators, state-of-the-art and perspectives of each of the referred issues.

scholarly journals FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yanping Long ◽  
Zhijian Liu ◽  
Jinbu Jia ◽  
Weipeng Mo ◽  
Liang Fang ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Walls ◽  
Large Scale ◽  
Full Length ◽  
Cell Level ◽  
Root Cells ◽  
Rna Profiling ◽  
Different Types ◽  
Long Read ◽  
Single Nucleus

AbstractThe broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

scholarly journals MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Julien Masliah-Planchon ◽  
Elodie Girard ◽  
Philipp Euskirchen ◽  
Christine Bourneix ◽  
Delphine Lequin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Nanopore Sequencing ◽  
Molecular Subgroup ◽  
Group Assignment ◽  
Group 4 ◽  
Methylation Assay ◽  
Tumor Group ◽  
Long Read ◽  
Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

scholarly journals Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Error Detection ◽  
Single Cells ◽  
Fusion Transcript ◽  
Building Blocks ◽  
Myeloma Cell ◽  
Nanopore Sequencing ◽  
Long Read ◽  
Unique Molecular Identifier ◽  
Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

scholarly journals Bio-Flocculation Property Analyses of Oleaginous Microalgae Auxenochlorella protothecoides UTEX 2341

2021 ◽  
Vol 13 (5) ◽  
pp. 2885
Author(s):  
Jinyu Li ◽  
Baozhen Li ◽  
Jinshui Yang
Keyword(s):  
Large Scale ◽  
Municipal Wastewater ◽  
Biofuel Production ◽  
Microalgae Harvesting ◽  
Flocculation Efficiency ◽  
Oleaginous Microalgae ◽  
Freeze Dried ◽  
Flocculation Ability ◽  
Powder Samples ◽  
Auxenochlorella Protothecoides

The bio-flocculation ability of UTEX 2341 was studied for the purpose of improving microalgae harvesting efficiency to cut the high cost of biofuel production. The algae cells of UTEX 2341 cultured under heterotrophic and municipal wastewater conditions were found to have better self-flocculation ability, with flocculation rates of 92% and 85% at 2 h, respectively. Moreover, the flocculation rates of 16 freeze-dried microalgae powder samples cultured under different stress conditions were 0~72% with an algae powder dosage of 35 mg L−1. The flocculation efficiency of DIM, DCd1, DT28, and L6S was stable under different pH of 3~9 and temperatures of 15~50 °C. For samples of IM, LCd0.6, LMn2, and LZn2, the flocculation efficiency decreased or increased respectively with increased pH or temperatures. Though the flocculation properties of the eight samples showed wide differences, their flocculant compositions were almost the same with unknown components occupying large proportions. More studies needed to be further carried out to reveal the flocculation mechanisms and analyze the flocculation abilities in practical application, which would be conducive to future large-scale application of the bio-flocculation method and also cost reduction.

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