PacBio Single-Molecule Long-Read Sequencing Provides New Light on the Complexity of Full-Length Transcripts in Cattle.
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ABSTRACT:
SUBMITTER: Chang T
PROVIDER: S-EPMC8437344 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Carex L., a grass genus commonly known as sedges, is distributed worldwide and contributes constructively to turf management, forage production, and ecological conservation. The development of next-generation sequencing (NGS) technologies has considerably improved our understanding of transcriptome complexity of Carex L. and provided a valuable genetic reference. However, the current transcriptome is not satisfactory mainly because of the enormous difficulty in obtaining full-length transcripts. RESULTS:In this study, we employed PacBio single-molecule long-read sequencing (SMRT) technology for whole-transcriptome profiling in Carex breviculmis. We generated 60,353 high-confidence non-redundant transcripts with an average length of 2302-bp. A total of 3588 alternative splicing events, and 1273 long non-coding RNAs were identified. Furthermore, 40,347 complete coding sequences were predicted, providing an informative reference transcriptome. In addition, the transcriptional regulation mechanism of C. breviculmis in response to shade stress was further explored by mapping the NGS data to the reference transcriptome constructed by SMRT sequencing. CONCLUSIONS:This study provided a full-length reference transcriptome of C. breviculmis using the SMRT sequencing method for the first time. The transcriptome atlas obtained will not only facilitate future functional genomics studies but also pave the way for further selective and genic engineering breeding projects for C. breviculmis.
| S-EPMC6821003 | biostudies-literature
Project description:BackgroundThe pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms.ResultsHerein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment.ConclusionsOur results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.
| S-EPMC7359550 | biostudies-literature
Project description:Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae. arabicum gene annotation using 294 RNA-seq libraries and 136 307 full-length PacBio Iso-seq transcripts, increasing BUSCO completeness by 11.6% and featuring 5606 additional genes. Analysis of orthologs showed a lower number of genes in Ae. arabicum than in other Brassicaceae, which could be partially explained by loss of homeologs derived from the At-α polyploidization event and by a lower occurrence of tandem duplications after divergence of Aethionema from the other Brassicaceae. Benchmarking of MADS-box genes identified orthologs of FUL and AGL79 not found in previous versions. Analysis of full-length transcripts related to ABA-mediated seed dormancy discovered a conserved isoform of PIF6-β and antisense transcripts in ABI3, ABI4 and DOG1, among other cases found of different alternative splicing between Turkey and Cyprus ecotypes. The presented data allow alternative splicing mining and proposition of numerous hypotheses to research evolution and functional genomics. Annotation data and sequences are available at the Ae. arabicum DB (https://plantcode.online.uni-marburg.de/aetar_db).
| S-EPMC8641386 | biostudies-literature
Project description:The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.
| S-EPMC6941099 | biostudies-literature
Project description:Chloroplast and mitochondrial DNA (cpDNA and mtDNA) are apart from nuclear DNA (nuDNA) in a eukaryotic cell. The transcription system of chloroplasts differs from those of mitochondria and eukaryotes. In contrast to nuDNA and animal mtDNA, the transcription of cpDNA is still not well understood, primarily due to the unresolved identification of transcription initiation sites (TISs) and transcription termination sites (TTSs) on the genome scale. In the present study, we characterized the transcription of chloroplast (cp) genes with greater accuracy and comprehensive information using PacBio full-length transcriptome data from Arabidopsis thaliana. The major findings included the discovery of four types of artifacts, the validation and correction of cp gene annotations, the exact identification of TISs that start with G, and the discovery of polyA-like sites as TTSs. Notably, we proposed a new model to explain cp transcription initiation and termination at the whole-genome level. Four types of artifacts, degraded RNAs and splicing intermediates deserve the attention from researchers working with PacBio full-length transcriptome data, as these contaminant sequences can lead to incorrect downstream analysis. Cp transcription initiates at multiple promoters and terminates at polyA-like sites. Our study provides new insights into cp transcription and new clues to study the evolution of promoters, TISs, TTSs and polyA tails of eukaryotic genes.
| S-EPMC10228390 | biostudies-literature
Project description:Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis. We present an integrated computational toolkit named TAGET for Iso-seq full-length transcript data analyses, including transcript alignment, annotation, gene fusion detection, and quantification analyses such as differential expression gene analysis and differential isoform usage analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.
| S-EPMC10518008 | biostudies-literature
Project description:The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.
| S-EPMC4915659 | biostudies-literature
Project description:BackgroundValsa canker is a serious disease in the stem of Malus sieversii, caused by Valsa mali. However, little is known about the global response mechanism in M. sieversii to V. mali infection.ResultsPhytohormone jasmonic acid (JA) and salicylic acid (SA) profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that the JA was initially produced to respond to the necrotrophic pathogen V. mali infection at the early response stage, then get synergistically transduced with SA to respond at the late response stage. Furthermore, we adopted Pacific Biosciences (PacBio) full-length sequencing to identify differentially expressed transcripts (DETs) during the canker response stage. We obtained 52,538 full-length transcripts, of which 8139 were DETs. Total 1336 lncRNAs, 23,737 alternative polyadenylation (APA) sites and 3780 putative transcription factors (TFs) were identified. Additionally, functional annotation analysis of DETs indicated that the wild apple response to the infection of V. mali involves plant-pathogen interaction, plant hormone signal transduction, flavonoid biosynthesis, and phenylpropanoid biosynthesis. The co-expression network of the differentially expressed TFs revealed 264 candidate TF transcripts. Among these candidates, the WRKY family was the most abundant. The MsWRKY7 and MsWRKY33 were highly correlated at the early response stage, and MsWRKY6, MsWRKY7, MsWRKY19, MsWRKY33, MsWRKY40, MsWRKY45, MsWRKY51, MsWRKY61, MsWRKY75 were highly correlated at the late stage.ConclusionsThe full-length transcriptomic analysis revealed a series of immune responsive events in M. sieversii in response to V. mali infection. The phytohormone signal pathway regulatory played an important role in the response stage. Additionally, the enriched disease resistance pathways and differentially expressed TFs dynamics collectively contributed to the immune response. This study provides valuable insights into a dynamic response in M. sieversii upon the necrotrophic pathogen V. mali infection, facilitates understanding of response mechanisms to canker disease for apple, and provides supports in the identification of potential resistance genes in M. sieversii.
| S-EPMC7809858 | biostudies-literature
Project description:Polyploidization contributes to the complexity of gene expression, resulting in numerous related but different transcripts. This study explored the transcriptome diversity and complexity of the tetraploid Arabica coffee (Coffea arabica) bean. Long-read sequencing (LRS) by Pacbio Isoform sequencing (Iso-seq) was used to obtain full-length transcripts without the difficulty and uncertainty of assembly required for reads from short-read technologies. The tetraploid transcriptome was annotated and compared with data from the sub-genome progenitors. Caffeine and sucrose genes were targeted for case analysis. An isoform-level tetraploid coffee bean reference transcriptome with 95 995 distinct transcripts (average 3236 bp) was obtained. A total of 88 715 sequences (92.42%) were annotated with BLASTx against NCBI non-redundant plant proteins, including 34 719 high-quality annotations. Further BLASTn analysis against NCBI non-redundant nucleotide sequences, Coffea canephora coding sequences with UTR, C. arabica ESTs, and Rfam resulted in 1213 sequences without hits, were potential novel genes in coffee. Longer UTRs were captured, especially in the 5΄UTRs, facilitating the identification of upstream open reading frames. The LRS also revealed more and longer transcript variants in key caffeine and sucrose metabolism genes from this polyploid genome. Long sequences (>10 kilo base) were poorly annotated. LRS technology shows the limitation of previous studies. It provides an important tool to produce a reference transcriptome including more of the diversity of full-length transcripts to help understand the biology and support the genetic improvement of polyploid species such as coffee.
| S-EPMC5737654 | biostudies-literature
Project description:BackgroundSwitchgrass (Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts.ResultsWe present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon-intron structures.ConclusionsOverall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass.
| S-EPMC6009963 | biostudies-literature