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      Full-Length Transcriptome Sequencing and the Discovery of New Transcripts in the Unfertilized Eggs of Zebrafish ( Danio rerio)

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          Abstract

          Understanding early gene expression in zebrafish embryos is a prerequisite for developmental biology research. In this study, 1,629,447 polymerase reads were obtained from the unfertilized eggs of zebrafish via full-length transcriptome sequencing using the PacBio RS II platform first. Then, 102,920 unique isoforms were obtained by correction, clustering and comparison with the zebrafish genome. 12,782 genes in the genome were captured, accounting for 39.71% of the all annotated genes. Approximately 62.27% of the 12,782 genes have been alternatively spliced. GO and KEGG annotations revealed that the unfertilized eggs primarily stored genes that participate in RNA processing and nuclear protein complex composition. According to this PacBio data that aligned with the genome, 3,970 fusion genes, 819 ncRNAs, and 84 new transcripts were predicted. Illumina RNA-seq and RT-qPCR detection found that the expression of two new transcripts, PB.5289.1 and PB.10209.1, were significantly up-regulated at the 2-cell stage and down-regulated rapidly thereafter, suggesting their involvement in minor ZGA during early embryonic development. This study indicated that the unfertilized eggs of zebrafish may have retained genes directly related to cell division and development to initiate the subsequent development in a limited space and time. On the other hand, NTRs or new transcriptome regions in the genome were discovered, which provided new clues regarding ZGA of MZT during early embryonic development in fish.

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          Most cited references 31

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          Alternative Isoform Regulation in Human Tissue Transcriptomes

          Through alternative processing of pre-mRNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes based on deep sequencing of cDNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analysis of mappings of sequence reads to exon-exon junctions indicated that 92-94% of human genes undergo alternative splicing (AS), ∼86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that a majority of AS and of alternative cleavage and polyadenylation (APA) events vary between tissues, while variation between individuals was ∼2- to 3-fold less common. Extreme or ‘switch-like’ regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full-length open reading frames. Patterns of AS and APA were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 3′ UTRs suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.
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            Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.

            We carried out the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice junctions were detected in approximately 20% of multiexon genes, many of which are tissue specific. By combining mRNA-Seq and EST-cDNA sequence data, we estimate that transcripts from approximately 95% of multiexon genes undergo alternative splicing and that there are approximately 100,000 intermediate- to high-abundance alternative splicing events in major human tissues. From a comparison with quantitative alternative splicing microarray profiling data, we also show that mRNA-Seq data provide reliable measurements for exon inclusion levels.
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              Real-time DNA sequencing from single polymerase molecules.

              We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                23 March 2019
                June 2019
                : 9
                : 6
                : 1831-1838
                Affiliations
                [* ]State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
                []University of Chinese Academy of Sciences, Beijing 100049, China
                Author notes
                [1]

                These authors contributed equally in this work.

                [2 ]Corresponding authors: E-mail: huangrong@ 123456ihb.ac.cn , wangyp@ 123456ihb.ac.cn
                Article
                GGG_200997
                10.1534/g3.119.200997
                6553537
                30872328
                Copyright © 2019 Mehjabin et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Figures: 3, Tables: 1, Equations: 0, References: 31, Pages: 8
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                Investigations

                Genetics

                zebrafish (danio rerio), rna-seq, unfertilized egg, full-length transcriptome sequencing

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