Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal…
The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various…
Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for…
To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant…
In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless…
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.…
Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts,…
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…
This tutorial provides an overview of the Isoform Sequence (Iso-Seq) analysis application. The Iso-Seq application provides reads that span entire transcript isoforms, from the 5′ end to the 3′ poly A-tail. Generation of accurate, full-length transcript sequences greatly simplifies analysis by eliminating the need for transcript reconstruction to infer isoforms using error-prone assembly of short RNA-seq reads. This tutorial covers features of SMRT Link v6.0.0.
In this PacBio User Group Meeting presentation, PacBio scientist Kristin Mars speaks about recent updates, such as the single-day library prep that’s now possible with the Iso-Seq Express workflow. She also notes that one SMRT Cell 8M is sufficient for most Iso-Seq experiments for whole transcriptome sequencing at an affordable price.
In this webinar, Kristin Mars, Sequencing Specialist, PacBio, presents an introduction to PacBio’s technology and its applications followed by a panel discussion among sequencing experts. The panel discussion addresses such things as what long reads are and how are they useful, what differentiates PacBio long-read sequencing from other technologies, and the applications PacBio offers and how they can benefit scientific research.
Winston Timp from Johns Hopkins University studies the metabolism of hummingbirds, which sustain the highest metabolic rates among all vertebrates. Notably, hummingbirds can switch rapidly between a fuel of lipids to newly ingested sugars. This remarkable metabolism is supported by enzymes which operate at the extreme limit of catalytic efficiency. Understanding the molecular basis of enzymatic action will provide a foundation enabling rational engineering of metabolic circuits in other systems. To do this, Dr. Timp and his team generated a de novo transcriptome of the hummingbird liver using the Iso-Seq method. Characterization of the resulting protein coding sequences provides clues…
In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains an overview of several applications: whole-genome sequencing for de novo assembly; transcript isoform sequencing (Iso-Seq) method for genome annotation; targeted sequencing solutions; and metagenomics and microbial interactions. High-level workflows and best practices are discussed for key applications.
PacBio 2014 User Group Meeting Presentation Slides: Alisha Holloway of the Gladstone Institutes presented on the use of isoform sequencing (Iso-Seq) to improve the annotation of the chicken genome as a model reference for cardiovascular research.
Advances in RNA sequencing have accelerated our understanding of the transcriptome, however isoform discovery remains challenging due to short read lengths. The Iso-Seq Application provides a new alternative to sequence full-length cDNA libraries using long reads from the PacBio RS II. Identification of long and often rare isoforms is demonstrated with rat heart and lung RNA prepared using the Clontech® SMARTer® cDNA preparation kit, followed by agarose-gel size selection in fractions of 1-2 kb, 2-3 kb and 3-6 kb. For each tissue, 1.8 and 1.2 million reads were obtained from 32 and 26 SMRT Cells, respectively. Filtering for reads with…