PacBio Iso-Seq Research Articles

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Full-length transcriptome sequencing and comparative transcriptome analysis of Eriocheir sinensis in response to infection by the microsporidian Hepatospora eriocheir.

As a new generation of high-throughput sequencing technology, PacBio Iso-Seq technology (Iso-Seq) provides a better alternative sequencing method for the acquisition of full-length unigenes. In this study, a total of 22.27 gigabyte (Gb) subread bases and 128,614 non-redundant unigenes (mean length: 2,324 bp) were obtained from six main tissues of Eriocheir sinensis including the heart, nerve, intestine, muscle, gills and hepatopancreas. In addition, 74,732 unigenes were mapped to at least one of the following databases: Non-Redundant Protein Sequence Database (NR), Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), KEGG Orthology (KO) and Protein family (Pfam). In addition, 6696 transcription factors (TFs), 28,458 long non-coding RNAs (lncRNAs) and 94,230 mRNA-miRNA pairs were identified. Hepatospora eriocheir is the primary pathogen of E. sinensis and can cause hepatopancreatic necrosis disease (HPND); the intestine is the main target tissue. Here, we attempted to identify the key genes related to H. eriocheir infection in the intestines of E. sinensis. By combining Iso-Seq and Illumina RNA-seq analysis, we identified a total of 12,708 differentially expressed unigenes (DEUs; 6,696 upregulated and 6,012 downregulated) in the crab intestine following infection with H. eriocheir. Based on the biological analysis of these DEUs, several key processes were identified, including energy metabolism-related pathways, cell apoptosis and innate immune-related pathways. Twelve selected genes from these DEUs were subsequently verified by quantitative real-time PCR (qRT-PCR) analysis. Our findings enhance our understanding of the E. sinensis transcriptome and the specific association between E. sinensis and H. eriocheir infection.

Transcriptome diversity assessment of Gossypium arboreum (FDH228) leaves under control, drought and whitefly infestation using PacBio long reads

Alternative splicing (AS) and alternative polyadenylation (APA) are common mechanisms in eukaryotes to increase the complexity of transcriptomes and subsequently proteomes. Analysis of long reads transcriptomics data can result in the discovery of novel transcripts, splice sites, AS or APA events. Gossypium arboreum is an important cultivated cotton species and a putative contributor of the A sub-genome to the modern tetraploid cotton; and inherently tolerant to several biotic and abiotic stresses. Specifically, its variety ‘FDH228′ is considered to be an important resistance source. In this study, we sequenced the G. arboreum (var. FDH228) transcriptome using PacBio IsoSeq and illumina short read sequencing under three different conditions i.e. untreated/healthy, treated with biotic stress through whitefly infestation, and treated with abiotic stress via water deprivation, for the discovery and surveying of canonical and non-canonical AS, APA and transcript fusion events. We were able to obtain 15,419 unique transcripts from all samples representing 11,343 genes, out of which 10,832 were annotated and 520 were novel with respect to the published reference genome. These transcripts were grouped into different structural categories including 60 Antisense, 11,959 having a full-splice match, 999 with incomplete-splice match, 30 fusion transcripts, 177 genic, 479 intergenic, 771 novels in the catalog, and 944 Novel but not found in the catalog. Subsequently, randomly selected candidate transcripts were experimentally validated using qRT-PCR. Our comprehensive identification of canonical and non-canonical splicing events, and novel and fusion transcripts aids in the understanding of the resistance mechanisms for this specific germplasm.

Transcript- and annotation-guided genome assembly of the European starling.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second, North American, short-read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species-specific transcript mapping and gene annotation revealed good gene-level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species.

The first long-read nuclear genome assembly of Oryza australiensis, a wild rice from northern Australia

Oryza australiensis is a wild rice native to monsoonal northern Australia. The International Oryza Map Alignment Project emphasises its significance as the sole representative of the EE genome clade. Assembly of the O. australiensis genome has previously been challenging due to its high Long Terminal Repeat (LTR) retrotransposon (RT) content. Oxford Nanopore long reads were combined with Illumina short reads to generate a high-quality ~ 858 Mbp genome assembly within 850 contigs with 46× long read coverage. Reference-guided scaffolding increased genome contiguity, placing 88.2% of contigs into 12 pseudomolecules. After alignment to the Oryza sativa cv. Nipponbare genome, we observed several structural variations. PacBio Iso-Seq data were generated for five distinct tissues to improve the functional annotation of 34,587 protein-coding genes and 42,329 transcripts. We also report SNV numbers for three additional O. australiensis genotypes based on Illumina re-sequencing. Although genetic similarity reflected geographical separation, the density of SNVs also correlated with our previous report on variations in salinity tolerance. This genome re-confirms the genetic remoteness of the O. australiensis lineage within the O. officinalis genome complex. Assembly of a high-quality genome for O. australiensis provides an important resource for the discovery of critical genes involved in development and stress tolerance.

Transcript Complexity and New Insights of Restorer Line in CMS-D8 Cotton Through Full-Length Transcriptomic Analysis.

Hybrid utilization has proficiently increased crop production worldwide. The cytoplasmic male sterility (CMS) system has emerged as an efficient tool for commercial hybrid cotton seed production. The restorer line with dominant Rf2 gene can restore the fertility of the CMS-D8 sterile line. However, the molecular mechanism of fertility restoration remains unclear in CMS-D8 cotton that limits wider utilization of three-line hybrid breeding. In our study, the Pacific Biosciences (PacBio) Iso-Seq technology was applied to understand fertility restoration mechanism of CMS-D8 cotton. In total, 228,106 full-length non-chimeric transcriptome sequences were obtained from anthers of developing flowering buds. The analysis results identified 3,174 novel isoforms, 2,597 novel gene loci, 652 long non-coding RNAs predicted from novel isoforms, 7,234 alternative splicing events, 114 fusion transcripts, and 1,667 genes with alternative polyadenylation. Specially, two novel genes associated with restoration function, Ghir_D05.742.1 and m64033_190821_201011/21103726/ccs were identified and showed significant higher levels of expression in restorer line than sterile and maintainer lines. Our comparative full-length transcriptome analysis provides new insights into the molecular function of Rf2 fertility restorer gene. The results of this study offer a platform for fertility restoration candidate gene discovery in CMS-D8 cotton.

Full-Length Transcriptomics Reveals Complex Molecular Mechanism of Salt Tolerance in Bromus inermis L.

Bromus inermis L. (commonly known as smooth bromegrass) is a grass species with high nutritional value, great palatability, cold tolerance, and grazing resistance, which has been widely cultivated for pasture and sand fixation in northern and northwestern China. Salt stress is a main environmental factor limiting growth and production of smooth bromegrass. In this study, we performed PacBio Iso-Seq to construct the first full-length transcriptome database for smooth bromegrass under 300 mM NaCl treatment at different time points. Third-generation full-length transcriptome sequencing yielded 19.67 G polymerase read bases, which were assembled into 355,836 full-length transcripts with an average length of 2,542 bp. A total of 116,578 differentially expressed genes were obtained by comparing the results of third-generation sequencing and second-generation sequencing. GO and KEGG enrichment analyses revealed that multiple pathways were differently activated in leaves and roots. In particular, a number of genes participating in the molecular network of plant signal perception, signal transduction, transcription regulation, antioxidant defense, and ion regulation were affected by NaCl treatment. In particular, the CBL-CIPK, MAPK, ABA signaling network, and SOS core regulatory pathways of Ca2+ signal transduction were activated to regulate salt stress response. In addition, the expression patterns of 10 salt-responsive genes were validated by quantitative real-time PCR, which were consistent with those detected by RNA-Seq. Our results reveal the molecular regulation of smooth bromegrass in response to salt stress, which are important for further investigation of critical salt responsive genes and molecular breeding of salt-tolerant smooth bromegrass.

Divergence of two cultivated allotetraploid cottons unveiled by single-molecule long-read expression sequencing

Allotetraploid Gossypium hirsutum and G. barbadense are two major cultivated cotton species that have different fiber traits. Research into the molecular basis of those differences is limited by a lack of accurate, complete transcript profiles. Here we adopted joint PacBio Iso-seq and RNA-seq to investigate genome-wide transcript profiles and uncover differences in the expression between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. We obtained 178,867 full-length transcripts covering 60.0% of gene loci in the TM-1 reference annotation, including 7846 transcription factors and 3122 lncRNAs. Moreover, we identified 304 and 610 high-confidence (HC) species-specific transcripts in TM-1 and Hai7124, respectively, along with 4156 and 4381 tissue-specific transcripts. Our results update the reference genome annotation and also provide potential resources for studying the molecular mechanisms of fiber development. We are thus able to promote improvements in cotton fiber quality. • Produced 178,867 full-length transcripts from 10 Gossypium hirsutum and G. barbadense tissues using single-molecule sequencing technology. • By contrast, species-specific and tissue-specific transcripts were identified. • Helps to uncover the molecular mechanisms underlying fiber differences at the transcript level in cotton.

Full-Length Transcriptome Sequencing and Comparative Transcriptomic Analyses Provide Comprehensive Insight Into Molecular Mechanisms of Cellulose and Lignin Biosynthesis in Cunninghamia lanceolata.

Cunninghamia lanceolata is an essential timber species that provide 20%–30% raw materials for China’s timber industry. Although a few transcriptomes have been published in C. lanceolata, full-length mRNA transcripts and regulatory mechanisms behind the cellulose and lignin biosynthesis have not been thoroughly investigated. Here, PacBio Iso-seq and RNA-seq analyses were adapted to identify the full-length and differentially expressed transcripts along a developmental gradient from apex to base of C. lanceolata shoots. A total of 48,846 high-quality full-length transcripts were obtained, of which 88.0% are completed transcriptome based on benchmarking universal single-copy orthologs (BUSCO) assessment. Along stem developmental gradient, 18,714 differentially expressed genes (DEGs) were detected. Further, 28 and 125 DEGs were identified as enzyme-coding genes of cellulose and lignin biosynthesis, respectively. Moreover, 57 transcription factors (TFs), including MYB and NAC, were identified to be involved in the regulatory network of cellulose and lignin biosynthesis through weighted gene co-expression network analysis (WGCNA). These TFs are composed of a comparable regulatory network of secondary cell wall formation in angiosperms, revealing a similar mechanism may exist in gymnosperms. Further, through qRT-PCR, we also investigated eight specific TFs involved in compression wood formation. Our findings provide a comprehensive and valuable source for molecular genetics breeding of C. lanceolata and will be beneficial for molecular-assisted selection.

Full-Length Transcriptome Analysis of the Halophyte Nitraria sibirica Pall.

Background: Nitraria sibirica Pall. is one of the pioneer tree species in saline–alkali areas due to its extreme salt tolerance. However, the lack of information on its genome limits the further exploration of the molecular mechanisms in N. sibirica under salt stress. Methods: In this study, we used single-molecule real-time (SMRT) technology based on the PacBio Iso-Seq platform to obtain transcriptome data from N. sibirica under salt treatment for the first time, which is helpful for our in-depth analysis of the salt tolerance and molecular characteristics of N. sibirica. Results: Our results suggested that a total of 234,508 circular consensus sequences (CCSs) with a mean read length of 2121 bp were obtained from the 19.26 Gb raw data. Furthermore, based on transcript cluster analysis, 93,713 consensus isoforms were obtained, including 92,116 high-quality isoforms. After removing redundant sequences, 49,240 non-redundant transcripts were obtained from high-quality isoforms. A total of 37,261 SSRs, 1816 LncRNAs and 47,314 CDSs, of which 40,160 carried complete ORFs, were obtained. Based on our transcriptome data, we also analyzed the coding genes of H+-PPase, and the results of both bioinformatics and functional analyses indicated that the gene prediction via full-length transcripts obtained by SMRT technology is reliable and effective. In summary, our research data obtained by SMRT technology provides more reliable and accurate information for the further analysis of the regulatory network and molecular mechanism of N. sibirica under salt stress.

Full-Length Transcriptome Sequencing-Based Analysis of Pinus sylvestris var. mongolica in Response to Sirex noctilio Venom

Simple SummarySirex noctilio, as a devastating international forestry quarantine pest whose venom can cause a series of physiological changes in the host plants, such as needle wilting, yellowing, decreased transpiration rate and increased respiration rate, etc. In this study, a full-length reference transcript of Pinus sylvestris var. mongolica was constructed by combining second- and third-generation transcriptome sequencing technologies. We also identified the specific expression genes and transcription factors of P. sylvestris var. mongolica under S. noctilio venom and wounding stress. S. noctilio venom mainly induced the expression of genes related to ROS, GAPDH and GPX, and mechanical damage mainly induced the photosynthesis−related genes. The results provide a better understanding of the molecular regulation of pine trees in response to S. noctilio venom.Sirex noctilio is a major international quarantine pest that recently emerged in northeast China to specifically invade conifers. During female oviposition, venom is injected into the host together with its symbiotic fungus to alter the normal Pinus physiology and weaken or even kill the tree. In China, the Mongolian pine (Pinus sylvestris var. mongolica), an important wind-proof and sand-fixing species, is the unique host of S. noctilio. To explore the interplay between S. noctilio venom and Mongolian pine, we performed a transcriptome comparative analysis of a 10-year-old Mongolian pine after wounding and inoculation with S. noctilio venom. The analysis was performed at 12 h, 24 h and 72 h. PacBio ISO-seq was used and integrated with RNA-seq to construct an accurate full-length transcriptomic database. We obtained 52,963 high-precision unigenes, consisting of 48,654 (91.86%) unigenes that were BLASTed to known sequences in the public database and 4309 unigenes without any annotation information, which were presumed to be new genes. The number of differentially expressed genes (DEGs) increased with the treatment time, and the DEGs were most abundant at 72 h. A total of 706 inoculation-specific DEGs (475 upregulated and 231 downregulated) and 387 wounding-specific DEGs (183 upregulated and 204 downregulated) were identified compared with the control. Under venom stress, we identified 6 DEGs associated with reactive oxygen species (ROS) and 20 resistance genes in Mongolian pine. Overall, 52 transcription factors (TFs) were found under venom stress, 45 of which belonged to the AP2/ERF TF family and were upregulated. A total of 13 genes related to the photosystem, 3 genes related photo-regulation, and 9 TFs were identified under wounding stress. In conclusion, several novel putative genes were found in Mongolian pine by PacBio ISO seq. Meanwhile, we also identified various genes that were resistant to S. noctilio venom, such as GAPDH, GPX, CAT, FL2, CERK1, and HSP83A, etc.

An Analysis Pipeline for Identification of RNA Modification, Alternative Splicing and Polyadenylation Using Third Generation Sequencing

Nanopore sequencing based on Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) have shown great potential in detecting post-transcriptional regulation. Direct RNA sequencing (DRS) has the advantages in capturing RNA modification due to without PCR amplification which is the limitation of the next-generation sequencing (NGS). Here, we provide a comprehensive computational procedure for the quantification of RNA modification in single-base resolution based on DRS data. Moreover, we also provide procedure on the identification of alternative splicing (AS) and alternative polyadenylation (APA) based on both DRS and PacBio Iso-Seq data. The entire step was based on two packages (Nanom6A and PRAPI), which were based on Python language on Linux system.Graphical abstract: The flowchart for identification of RNA modification, alternative splicing and alternative polyadenylation based on third generation sequencing technology., [摘要]基于 Oxford Nanopore Technologies (ONT) 和 Pacific 的纳米孔测序 BioSciences (PacBio) 单分子实时 (SMRT) 长读长异构体测序 (Iso-Seq) 在检测转录后调控方面显示出巨大潜力。直接 RNA测序 (DRS) 由于没有 PCR 扩增而在捕获 RNA 修饰方面具有优势，这是下一代测序(NGS)的限制。在这里，我们提供了一个全面的计算程序，用于基于 DRS 数据以单碱基分辨率量化 RNA 修饰。此外，我们还提供了基于 DRS 和 PacBio Iso-Seq 数据识别选择性剪接(AS)和选择性多聚腺苷酸化(APA)的程序。整个步骤基于两个包（Nanom6A 和 PRAPI），它们基于 Linux 系统上的 Python 语言。关键词： 纳米孔直接 RNA 测序，PacBio Iso-Seq，RNA 修饰， N 6 -甲基腺苷，选择性剪接，选择性多聚腺苷酸化图文摘要：基于第三代测序技术的RNA修饰、选择性剪接和选择性多聚腺苷酸化鉴定流程图。[背景]转录组为理解基因表达和生长发育的调控提供了深入的视角。 随着第三代测序技术的快速发展，基于长读长测序的数据在揭示转录后调控方面具有很大优势（ Smith et al ., 2019） 。转录后 调控被认为是一个复杂的生物学过程，在影响 RNA 的剪接、输出、定位和翻译方面发挥着重要作用（Keene，2007） 。例如，选择性多聚腺苷酸化 (APA) 在维持 RNA 的结构和稳定性方面起着至关重要的作用 （Di Giamartino等人，2011 年，Tian 和 Manley，2017 年） 。选择性剪接(AS) )根茎相关发育的调控 (Wang et al ., 2017)和激素反应(Zhang et al ., 2018) 。在与生物合成途径相关的关键基因中可以发现多种 AS 事件(Chen et al ., 2020) 。 PacBio Iso-Seq 可以在同种型范围内捕获全长 mRNA以分析 转录后处理事件，例如AS 和 APA （Rhoads 和 Au，2015 年） 。准确性和可靠性工具对于识别转录后事件很重要。短读平台上的AS base d可以被几个优秀的软件识别，如rMATS （Shen等人，2014 年）和 SUPPA2 （Trincado等人，2018 年） 。然而，由于转录组组装的限制，短读长平台在检测全长异构体方面表现出劣势（ Rhoads和 Au，2015 年） 。 TAPIS (Abdel-Ghany et al ., 2016)和 FLAIR (Tang et al ., 2020) 已被开发用于基于长读序列识别 AS 事件。然而，TAPIS 不提供转录后事件的定量分析（ Abdel -Ghany等，2016） 。 FLAIR 可以检测亚型，但缺少 APA 检测模块（Tang等 人。 , 2020) 。 因此，我们为AS 和 APA的识别和可视化提供了一个全面且用户友好的管道 (PRAPI) (Gao et al ., 2018) 。在这里，我们提供了使用基于 Iso-Seq 异构体和短读数据的 PRAPI 进行数据处理、可视化、识别 AS 和 APA 的详细程序。除了 PacBio 平台， Oxford Nanopore Technologies (ONT) 平台还应用了独特的测量方法，将 RNA 分子通过特殊孔隙的信息作为电信号保持 （Garalde等人，2018 年） ， 它可以识别包括N 6 -甲基腺苷 (m 6 A) (Linder et al ., 2015; Zhang et al ., 2019) 、假尿苷(Ψ) 和 2'-O-甲基化 (Nm)在内的 RNA 修饰 （贝吉克等人，2021） 基于天然 RNA 的单核苷酸分辨率。此外，Nanopore DRS 能够确定 RNA 二级结构(Aw et al ., 2021) ，并检测天然去折叠蛋白序列中的氨基酸(Hu et al ., 2021) 。基于 ONT 数据检测 RNA 修饰的方法已在包括Epinano在内的多个软件包中报告 （刘等人，2019） ， xPore （普拉坦瓦尼奇 等人，2021 年） ，MINES （洛伦兹等人，2020 年） ， NanoDoc (Ueda, 2021) 、Nanom6A (Gao et al ., 2021) 、 Tombo (斯托伊伯 等人，2017） 。 Epinano 、 xPore 、Nanom6A 和 MINES 可以实现单碱基分辨率的准确度。然而，由于依赖碱基调用错误， Epinano无法将 m 6 A 与其他修饰碱基（例如 m 1 A）区分开来（Liu et al ., 2019） 。此外， xPore需要 m 6 A writer 敲除线作为比较，并且可能由于其在 DRACH 基序中的强富集而存在偏差（Pratanwanich等人，2021） 。最后，MINES 仅检测到 4 个 RRACH 基序（AGACT、GGACA、GGACC 和 GGACT），而忽略了其他 8 个 RRACH 基序（Lorenz等人，2020） 。 Nanom6A提供了使用 Nanopore 直接 RNA 读数来鉴定定性和定量 m 6 A 的方法，基于XGBoost算法以单碱基分辨率修改，提供 m 6 A 的高精度全转录组鉴定、定量和序列上下文 (RRACH )修改（Gao et al ., 2021） 。在这项研究中，我们提供了详细的程序 定量精度高 以单碱基分辨率进行 RNA 修饰。简而言之，我们提供了基于 DRS 或 PacBio Iso-Seq 数据鉴定AS 、 APA 和 m 6 A的综合程序。用于 Nanom6A 和 PRAPI 分析的 Linux Bash Shell 和脚本现已在https://github.com/GuInNGS/NanoPrapi中提供 和https://github.com/Bio-protocol/Pipeline_for_Identification_m6A_AS_and_APA_with_Long_Read 。

Unveiling the Complexity of Red Clover (Trifolium pratense L.) Transcriptome and Transcriptional Regulation of Isoflavonoid Biosynthesis Using Integrated Long- and Short-Read RNAseq.

Red clover (Trifolium pratense L.) is used as forage and contains a high level of isoflavonoids. Although isoflavonoids in red clover were discovered a long time ago, the transcriptional regulation of isoflavonoid biosynthesis is virtually unknown because of the lack of accurate and comprehensive characterization of the transcriptome. Here, we used a combination of long-read (PacBio Iso-Seq) and short-read (Illumina) RNAseq sequencing to develop a more comprehensive full-length transcriptome in four tissues (root, stem, leaf, and flower) and to identify transcription factors possibly involved in isoflavonoid biosynthesis in red clover. Overall, we obtained 50,922 isoforms, including 19,860 known genes and 2817 novel isoforms based on the annotation of RefGen Tp_v2.0. We also found 1843 long non-coding RNAs, 1625 fusion genes, and 34,612 alternatively spliced events, with some transcript isoforms validated experimentally. A total of 16,734 differentially expressed genes were identified in the four tissues, including 43 isoflavonoid-biosynthesis-related genes, such as stem-specific expressed TpPAL, TpC4H, and Tp4CL and root-specific expressed TpCHS, TpCHI1, and TpIFS. Further, weighted gene co-expression network analysis and a targeted compound assay were combined to investigate the association between the isoflavonoid content and the transcription factors expression in the four tissues. Twelve transcription factors were identified as key genes for isoflavonoid biosynthesis. Among these transcription factors, the overexpression of TpMYB30 or TpRSM1-2 significantly increased the isoflavonoid content in tobacco. In particular, the glycitin was increased by 50–100 times in the plants overexpressing TpRSM1-2, in comparison to that in the WT plants. Our study provides a comprehensive and accurate annotation of the red clover transcriptome and candidate genes to improve isoflavonoid biosynthesis and accelerate research into molecular breeding in red clover or other crops.

Transcriptome profiling based on Illumina- and SMRT-based RNA-seq reveals circadian regulation of key pathways in flower bud development in walnut.

Flower bud development is a defining feature of walnut, which contributes to the kernel yield, yield stability, fruit quality and commodity value. However, little is known about the mechanism of the flower bud development in walnut. Here, the stages of walnut female flower bud development were divided into five period (P01-05) by using histological observation. They were further studied through PacBio Iso-Seq and RNA-seq analysis. Accordingly, we obtained 52,875 full-length transcripts, where 4,579 were new transcripts, 3,065 were novel genes, 1,437 were consensus lncRNAs and 20,813 were alternatively spliced isoforms. These transcripts greatly improved the current genome annotation and enhanced our understanding of the walnut transcriptome. Next, RNA sequencing of female flower buds at five periods revealed that circadian rhythm-plant was commonly enriched along with the flower bud developmental gradient. A total of 14 differentially expressed genes (DEGs) were identified, and six of them were confirmed by real-time quantitative analysis. Additionally, six and two differentially expressed clock genes were detected to be regulated by AS events and lncRNAs, respectively. All these detected plant circadian genes form a complex interconnected network to regulate the flower bud development. Thus, investigation of key genes associated with the circadian clock could clarify the process of flower bud development in walnut.

The Full-Length Transcriptome Provides New Insights Into the Transcript Complexity of Abdominal Adipose and Subcutaneous Adipose in Pekin Ducks.

Adipose tissues have a central role in organisms, and adipose content is a crucial economic trait of poultry. Pekin duck is an ideal model to study the mechanism of abdominal and subcutaneous adipose deposition for its high ability of adipose synthesis and deposition. Alternative splicing contributes to functional diversity in abdominal and subcutaneous adipose. However, there has been no systematic analysis of the dynamics of differential alternative splicing of abdominal and subcutaneous adipose in Pekin duck. In our study, the Pacific Biosciences (PacBio) Iso-Seq technology was applied to explore the transcriptional complexity of abdominal and subcutaneous adipose in Pekin ducks. In total, 143,931 and 111,337 full-length non-chimeric transcriptome sequences of abdominal and subcutaneous adipocytes were obtained from 41.78 GB raw data, respectively. These data led us to identify 19,212 long non-coding RNAs (lncRNAs) and 74,571 alternative splicing events. In addition, combined with the next-generation sequencing technology, we correlated the structure and function annotation with the differential expression profiles of abdominal and subcutaneous adipose transcripts. This study identified lots of novel alternative splicing events and major transcripts of transcription factors related to adipose synthesis. STAT3 was reported as a vital gene for adipogenesis, and we found that its major transcript is STAT3-1, which may play a considerable role in the process of adipose synthesis in Pekin duck. This study greatly increases our understanding of the gene models, genome annotations, genome structures, and the complexity and diversity of abdominal and subcutaneous adipose in Pekin duck. These data provide insights into the regulation of alternative splicing events, which form an essential part of transcript diversity during adipogenesis in poultry. The results of this study provide an invaluable resource for studying alternative splicing and tissue-specific expression.

Resolving the Microalgal Gene Landscape at the Strain Level: a Novel Hybrid Transcriptome of Emiliania huxleyi CCMP3266.

Microalgae are key ecological players with a complex evolutionary history. Genomic diversity, in addition to limited availability of high-quality genomes, challenge studies that aim to elucidate molecular mechanisms underlying microalgal ecophysiology. Here, we present a novel and comprehensive transcriptomic hybrid approach to generate a reference for genetic analyses and resolve the microalgal gene landscape at the strain level. The approach is demonstrated for a strain of the coccolithophore microalga Emiliania huxleyi, which is a species complex with considerable genome variability. The investigated strain is commonly studied as a model for algal-bacterial interactions and was therefore sequenced in the presence of bacteria to elicit the expression of interaction-relevant genes. We applied complementary PacBio Iso-Seq full-length cDNA and poly(A)-independent Illumina total RNA sequencing, which resulted in a de novo-assembled, near-complete hybrid transcriptome. In particular, hybrid sequencing improved the reconstruction of long transcripts and increased the recovery of full-length transcript isoforms. To use the resulting hybrid transcriptome as a reference for genetic analyses, we demonstrate a method that collapses the transcriptome into a genome-like data set, termed "synthetic genome" (sGenome). We used the sGenome as a reference to visually confirm the robustness of the CCMP3266 gene assembly, to conduct differential gene expression analysis, and to characterize novel E. huxleyi genes. The newly identified genes contribute to our understanding of E. huxleyi genome diversification and are predicted to play a role in microbial interactions. Our transcriptomic toolkit can be implemented in various microalgae to facilitate mechanistic studies on microalgal diversity and ecology. IMPORTANCE Microalgae are key players in the ecology and biogeochemistry of our oceans. Efforts to implement genomic and transcriptomic tools in laboratory studies involving microalgae suffer from the lack of published genomes. In the case of coccolithophore microalgae, the problem has long been recognized; the model species Emiliania huxleyi is a species complex with genomes composed of a core and a large variable portion. To study the role of the variable portion in niche adaptation, and specifically in microbial interactions, strain-specific genetic information is required. Here, we present a novel transcriptomic hybrid approach, and generated strain-specific genome-like information. We demonstrate our approach on an E. huxleyi strain that is cocultivated with bacteria. By constructing a "synthetic genome," we generated comprehensive gene annotations that enabled accurate analyses of gene expression patterns. Importantly, we unveiled novel genes in the variable portion of E. huxleyi that play putative roles in microbial interactions.

Transcriptome-wide characterization and functional analysis of Xyloglucan endo-transglycosylase/hydrolase (XTH) gene family of Salicornia europaea L. under salinity and drought stress

BackgroundSalicornia europaea is a halophyte that has a very pronounced salt tolerance. As a cell wall manipulating enzyme, xyloglucan endotransglycosylase/hydrolase (XTH) plays an important role in plant resistance to abiotic stress. However, no systematic study of the XTH gene family in S. europaea is well known. PacBio Iso-Seq transcriptome sequence data were used for bioinformatics and gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR).ResultsTranscriptome sequencing (PacBio Iso-Seq system) generated 16,465,671 sub-reads and after quality control of Iso-Seq, 29,520 isoforms were obtained with an average length of 2112 bp. A total of 24,869 unigenes, with 98% of which were obtained using coding sequences (CDSs), and 6398 possible transcription factors (TFs) were identified. Thirty-five (35) non-redundant potential SeXTH proteins were identified in S. europaea and categorized into group I/II and group III based on their genetic relatedness. Prediction of the conserved motif revealed that the DE(I/L/F/V)DF(I)EFLG domain was conserved in the S. europaea proteins and a potential N-linked glycosylation domain N(T)V(R/L/T/I)T(S/K/R/F/P)G was also located near the catalytic residues. All SeXTH genes exhibited discrete expression patterns in different tissues, at different times, and under different stresses. For example, 27 and 15 SeXTH genes were positively expressed under salt stress in shoots and roots at 200 mM NaCl in 24 h, and 34 SeXTH genes were also positively regulated under 48 h of drought stress in shoots and roots. This indicates their function in adaptation to salt and drought stress.ConclusionThe present study discovered SeXTH gene family traits that are potential stress resistance regulators in S. europaea, and this provides a basis for future functional diversity research.

First Annotated Genome of a Mandibulate Moth, Neomicropteryx cornuta, Generated Using PacBio HiFi Sequencing.

We provide a new, annotated genome assembly of Neomicropteryx cornuta, a species of the so-called mandibulate archaic moths (Lepidoptera: Micropterigidae). These moths belong to a lineage that is thought to have split from all other Lepidoptera more than 300 Ma and are consequently vital to understanding the early evolution of superorder Amphiesmenoptera, which contains the order Lepidoptera (butterflies and moths) and its sister order Trichoptera (caddisflies). Using PacBio HiFi sequencing reads, we assembled a highly contiguous genome with a contig N50 of nearly 17 Mb. The assembled genome length of 541,115,538 bp is about half the length of the largest published Amphiesmenoptera genome (Limnephilus lunatus, Trichoptera) and double the length of the smallest (Papilio polytes, Lepidoptera). We find high recovery of universal single copy orthologs with 98.1% of BUSCO genes present and provide a genome annotation of 15,643 genes aided by resolved isoforms from PacBio IsoSeq data. This high-quality genome assembly provides an important resource for studying ecological and evolutionary transitions in the early evolution of Amphiesmenoptera.

Unveiling the transcriptomic complexity of Miscanthus sinensis using a combination of PacBio long read- and Illumina short read sequencing platforms

BackgroundMiscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics suitable for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which considerably hampers further work on its biology and genetic improvement.ResultsIn this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gb clean data. An average of 15.75 Gb clean reads of each sample were obtained from the PacBio Iso-Seq system, which doubled the data size (6.68 Gb) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-Seq. Approximately 96 % of the final combined transcripts were mapped back to the Miscanthus genome, reflecting the high quality and coverage of our sequencing results. When comparing our data with genomes of four species of Andropogoneae, M. sinensis showed the closest relationship with sugarcane with up to 93 % mapping ratios, followed by sorghum with up to 80 % mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories.ConclusionsTaken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis transcriptomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.

The Landscapes of Full-Length Transcripts and Splice Isoforms as Well as Transposons Exonization in the Lepidopteran Model System, Bombyx mori.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.