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April 21, 2020  |  

Large Scale Profiling of Protein Isoforms Using Label-Free Quantitative Proteomics Revealed the Regulation of Nonsense-Mediated Decay in Moso Bamboo (Phyllostachys edulis).

Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.

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April 21, 2020  |  

Complete genome sequence of marine Bacillus sp. Y-01, isolated from the plastics contamination in the Yellow Sea

Plastics contamination in the environment has been an increasing ecological problem. Here we present the complete genome sequence of Bacillus sp. Y-01, isolated from plastic contamination samples in the Yellow Sea, which can utilize the polypropylene as the sole carbon and energy source. The strain has one circular chromosome of 5,130,901?bp in 8 contigs with a 38.24% GC content, consisting of 4996 protein-coding genes, 118 tRNA genes, as well as 40 rRNA operons as 5S-16S-23S rRNA. The complete genome sequence of Bacillus sp. Y-01 will provide useful genetic information to further detect the molecular mechanisms behind marine microplastics degradation.

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April 21, 2020  |  

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host. Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020  |  

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_IsoSeq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

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April 21, 2020  |  

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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April 21, 2020  |  

RNA-seq of HaHV-1-infected abalones reveals a common transcriptional signature of Malacoherpesviruses.

Haliotid herpesvirus-1 (HaHV-1) is the viral agent causative of abalone viral ganglioneuritis, a disease that has severely affected gastropod aquaculture. Although limited, the sequence similarity between HaHV-1 and Ostreid herpesvirus-1 supported the assignment of both viruses to Malacoherpesviridae, a Herpesvirales family distantly related with other viruses. In this study, we reported the first transcriptional data of HaHV-1, obtained from an experimental infection of Haliotis diversicolor supertexta. We also sequenced the genome draft of the Chinese HaHV-1 variant isolated in 2003 (HaHV-1-CN2003) by PacBio technology. Analysis of 13 million reads obtained from 3 RNA samples at 60?hours post injection (hpi) allowed the prediction of 51 new ORFs for a total of 117 viral genes and the identification of 207 variations from the reference genome, consisting in 135 Single Nucleotide Polymorphisms (SNPs) and 72 Insertions or Deletions (InDels). The pairing of genomic and transcriptomic data supported the identification of 60 additional SNPs, representing viral transcriptional variability and preferentially grouped in hotspots. The expression analysis of HaHV-1 ORFs revealed one putative secreted protein, two putative capsid proteins and a possible viral capsid protease as the most expressed genes and demonstrated highly synchronized viral expression patterns of the 3 infected animals at 60?hpi. Quantitative reverse transcription data of 37 viral genes supported the burst of viral transcription at 30 and 60?hpi during the 72?hours of the infection experiment, and allowed the distinction between early and late viral genes.

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April 21, 2020  |  

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, with a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed and many new generally shorter transcripts were detected by normalization. For the same input cDNA and the same data yield, the normalized library recovered more total transcript isoforms, number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ~1.25 kb, more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ~52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ~80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

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April 21, 2020  |  

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

Gossypium australe F. Mueller (2n?=?2x?=?26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

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April 21, 2020  |  

The transcriptome of Darwin’s bark spider silk glands predicts proteins contributing to dragline silk toughness.

Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials.

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