Assembly of full length 45s Ribosomal RNA sequences from Saccharum
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ABSTRACT:
PROVIDER: PRJEB38889 | ENA |
REPOSITORIES: ENA
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Project description:Here we describe CapTrap-Seq, an experimental workflow designed to address the problem of reduced transcript end detection by long-read RNA sequencing methods, especially at the 5' ends. We apply CapTrap-Seq to profile transcriptomes of the human heart and brain and we compared the obtained results with other library preparation approaches. CapTrap-Seq is a platform-agnostic method and here tested the method by using 3 different long-read sequencing platforms: MinION (ONT), Sequel (PacBaio) and Sequel II (PacBio).
2023-12-20 | E-MTAB-13063 | biostudies-arrayexpress
Project description:BackgroundAlthough extensive breeding efforts are ongoing in sugarcane (Saccharum officinarum L.), the average yield is far below the theoretical potential. Tillering is an important component of sugarcane yield, however, the molecular mechanism underlying tiller development is still elusive. The limited genomic data in sugarcane, particularly due to its complex and large genome, has hindered in-depth molecular studies.ResultsHerein, we generated full-length (FL) transcriptome from developing leaf and tiller bud samples based on PacBio Iso-Seq. In addition, we performed RNA-seq from tiller bud samples at three developmental stages (T0, T1 and T2) to uncover key genes and biological pathways involved in sugarcane tiller development. In total, 30,360 and 20,088 high-quality non-redundant isoforms were identified in leaf and tiller bud samples, respectively, representing 41,109 unique isoforms in sugarcane. Likewise, we identified 1063 and 1037 alternative splicing events identified in leaf and tiller bud samples, respectively. We predicted the presence of coding sequence for 40,343 isoforms, 98% of which was successfully annotated. Comparison with previous FL transcriptomes in sugarcane revealed 2963 unreported isoforms. In addition, we characterized 14,946 SSRs from 11,700 transcripts and 310 lncRNAs. By integrating RNA-seq with the FL transcriptome, 468 and 57 differentially expressed genes (DEG) were identified in T1vsT0 and T2vsT0, respectively. Strong up-regulation of several pyruvate phosphate dikinase and phosphoenolpyruvate carboxylase genes suggests enhanced carbon fixation and protein synthesis to facilitate tiller growth. Similarly, up-regulation of linoleate 9S-lipoxygenase and lipoxygenase genes in the linoleic acid metabolism pathway suggests high synthesis of key oxylipins involved in tiller growth and development.ConclusionsCollectively, we have enriched the genomic data available in sugarcane and provided candidate genes for manipulating tiller formation and development, towards productivity enhancement in sugarcane.
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Project description:The ribosome is a two-subunit, macromolecular machine composed of RNA and proteins that carries out the polymerization of α-amino acids into polypeptides. Efforts to engineer ribosomal RNA (rRNA) deepen our understanding of molecular translation and provide opportunities to expand the chemistry of life by creating ribosomes with altered properties. Toward these efforts, reverse transcription PCR (RT-PCR) of the entire 16S and 23S rRNAs, which make up the 30S small subunit and 50S large subunit, respectively, is important for isolating desired phenotypes. However, reverse transcription of rRNA is challenging due to extensive secondary structure and post-transcriptional modifications. One key challenge is that existing commercial kits for RT-PCR rely on reverse transcriptases that lack the extreme thermostability and processivity found in many commercial DNA polymerases, which can result in subpar performance on challenging templates. Here, we develop methods employing a synthetic thermostable reverse transcriptase (RTX) to enable and optimize RT-PCR of the complete Escherichia coli 16S and 23S rRNAs. We also characterize the error rate of RTX when traversing the various post-transcriptional modifications of the 23S rRNA. We anticipate that this work will facilitate efforts to study and characterize many naturally occurring long RNAs and to engineer the translation apparatus for synthetic biology.
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Project description:Full-length HLA class II sequences
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Project description:Blastocystis is a common food- and water-borne intestinal protist parasite of humans and many other animals. Blastocystis comprises multiple subtypes (STs) based on variability within the small subunit ribosomal (SSU rRNA) RNA gene. Though full-length reference sequences of the SSU rRNA gene are a current requirement to name a novel Blastocystis subtype, full-length reference sequences are not currently available for all subtypes. In the present study, Oxford Nanopore MinION long-read sequencing was employed to generate full-length SSU rRNA sequences for seven new Blastocystis subtypes for which no full-length references currently exist: ST21, ST23, ST24, ST25, ST26, ST27, and ST28. Phylogenetic analyses and pairwise distance matrixes were used to compare full-length and partial sequences of the two regions that are most commonly used for subtyping. Analyses included Blastocystis nucleotide sequences obtained in this study (ST21 and ST23-ST28) and existing subtypes for which full-length reference sequences were available (ST1-ST17 and ST29). The relationships and sequence variance between new and existing subtypes observed in analyses of different portions of the SSU rRNA gene are discussed. The full-length SSU rRNA reference sequences generated in this study provide essential new data to study and understand the relationships between the genetic complexity of Blastocystis and its host specificity, pathogenicity, and epidemiology.
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Project description:Diamond Blackfan anemia is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, often with associated physical abnormalities. Perturbations of the ribosome appear critically important to the development of DBA, as alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, presently only 50-60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide SNP array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9/51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A [Illumina platforms] A total of 68 samples were analyzed on Illumina Omni1_Quad or Illumina OmniExpress Genotyping bead arrays; 51 affected probands, 1 affected sibling, 1 unaffected sibling, and 15 parents. [NimbleGen platform] 5 samples where a deletion was identified by SNP array and one sample where no deletion was identified were run on Nimblegen Human CGH 3x720K Whole-Genome Exon-Focused arrays for confirmation.
2012-01-15 | E-GEOD-31575 | biostudies-arrayexpress