Menu

Auto Tag: Full-length mRNA

April 21, 2020  |  

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.

Read more
October 23, 2019  |  

Development of a Novel Reference Transcriptome for Scleractinian Coral Porites lutea Using Single-Molecule Long-Read Isoform Sequencing (Iso-Seq)

Elevation in seawater temperature associated with global climate change has caused coral bleaching problems and posed a significant threat to coral health and survival worldwide. Several studies have explored the effects of thermal stress on changes in gene expression levels of both coral hosts and their algal endosymbionts and provided evidences suggesting that corals could acclimatize to environmental stressors through differential regulation of their gene expression (Desalvo et al., 2008, 2010; Császár et al., 2009; Rodriguez-Lanetty et al., 2009; Polato et al., 2010; Meyer et al., 2011; Kenkel et al., 2013). Such information is crucial for understanding the adaptive capacity of the coral holobionts (Hughes et al., 2003). The availability of transcriptome data from a number of coral species and their associated Symbiodinium allows us to probe the molecular stress response of the organisms to heat stress (Traylor-Knowles et al., 2011; Moya et al., 2012; Kenkel et al., 2013; Shinzato et al., 2014; Kitchen et al., 2015; Anderson et al., 2016; Davies et al., 2016). Here, we report the first reference transcriptome for a scleractinian coral Porites lutea, one of the dominant reef-builders in the Indo-West Pacific (Yeemin et al., 2009). We applied both short-read Ion S5 RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate transcriptome sequences of P. lutea under normal and heat stress conditions. The key advantage of PacBio’s Iso-seq technology lies within its ability to capture full-length mRNA sequences. These full-length transcripts enable the identification of novel genes/isoforms and the detection of alternative splice variants, which have been shown to be overrepresented in stress responses (Iida et al., 2004; Reddy et al., 2013; Liu and Guo, 2017). We envision that this reference transcriptome will provide a coral research community a valuable resource for investigating changes in gene expression under various biotic/abiotic stress conditions.

Read more
September 22, 2019  |  

Alternative TSSs are co-regulated in single cells in the mouse brain.

Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS usage is a regulated process, and the correlation between two TSSs expressed in single cells of the same cell type is surprisingly high. Our findings indicate that TSSs generally are regulated by common factors rather than being independently regulated or stochastically expressed.© 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Read more
September 22, 2019  |  

neoantigenR: An annotation based pipeline for tumor neoantigen identification from sequencing data

Studies indicate that more than 90% of human genes are alternatively spliced, suggesting the complexity of the transcriptome assembly and analysis. The splicing process is often disrupted, resulting in both functional and non-functional end-products (Sveen et al. 2016) in many cancers. Harnessing the immune system to fight against malignant cancers carrying aberrantly mutated or spliced products is becoming a promising approach to cancer therapy. Advances in immune checkpoint blockade have elicited adaptive immune responses with promising clinical responses to treatments against human malignancies (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Emerging data suggest that recognition of patient-specific mutation-associated cancer antigens (i.e. from alternative splicing isoforms) may allow scientists to dissect the immune response in the activity of clinical immunotherapies (Schumacher and Schreiber 2015). The advent of high-throughput sequencing technology has provided a comprehensive view of both splicing aberrations and somatic mutations across a range of human malignancies, allowing for a deeper understanding of the interplay of various disease mechanisms. Meanwhile, studies show that the number of transcript isoforms reported to date may be limited by the short-read sequencing due to the inherit limitation of transcriptome reconstruction algorithms, whereas long-read sequencing is able to significantly improve the detection of alternative splicing variants since there is no need to assemble full-length transcripts from short reads. The analysis of these high-throughput long-read sequencing data may permit a systematic view of tumor specific peptide epitopes (also known as neoantigens) that could serve as targets for immunotherapy (Tumor Neoantigens in Personalized Cancer Immunotherapy 2017). Currently, there is no software pipeline available that can efficiently produce mutation-associated cancer antigens from raw high-throughput sequencing data on patient tumor DNA (The Problem with Neoantigen Prediction 2017). In addressing this issue, we introduce a R package that allows the discoveries of peptide epitope candidates, which are the tumor-specific peptide fragments containing potential functional neoantigens. These peptide epitopes consist of structure variants including insertion, deletions, alternative sequences, and peptides from nonsynonymous mutations. Analysis of these precursor candidates with widely used tools such as netMHC allows for the accurate in-silico prediction of neoantigens. The pipeline named neoantigeR is currently hosted in https://github.com/ICBI/neoantigeR.

Read more
September 22, 2019  |  

Improving PacBio long read accuracy by short read alignment.

The recent development of third generation sequencing (TGS) generates much longer reads than second generation sequencing (SGS) and thus provides a chance to solve problems that are difficult to study through SGS alone. However, higher raw read error rates are an intrinsic drawback in most TGS technologies. Here we present a computational method, LSC, to perform error correction of TGS long reads (LR) by SGS short reads (SR). Aiming to reduce the error rate in homopolymer runs in the main TGS platform, the PacBio® RS, LSC applies a homopolymer compression (HC) transformation strategy to increase the sensitivity of SR-LR alignment without scarifying alignment accuracy. We applied LSC to 100,000 PacBio long reads from human brain cerebellum RNA-seq data and 64 million single-end 75 bp reads from human brain RNA-seq data. The results show LSC can correct PacBio long reads to reduce the error rate by more than 3 folds. The improved accuracy greatly benefits many downstream analyses, such as directional gene isoform detection in RNA-seq study. Compared with another hybrid correction tool, LSC can achieve over double the sensitivity and similar specificity.

Read more
September 22, 2019  |  

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781?bp per gene in 82.473?Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals. Copyright © 2018. Published by Elsevier Inc.

Read more
September 22, 2019  |  

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

Parallel sequencing of a single cell’s genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ~3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

Read more
September 22, 2019  |  

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.

Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis.Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template.Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.

Read more
September 22, 2019  |  

cDNA library enrichment of full length transcripts for SMRT long read sequencing.

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.

Read more
September 22, 2019  |  

Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis.

RNA-sequencing (RNA-seq) is an essential technique for transcriptome studies, hundreds of analysis tools have been developed since it was debuted. Although recent efforts have attempted to assess the latest available tools, they have not evaluated the analysis workflows comprehensively to unleash the power within RNA-seq. Here we conduct an extensive study analysing a broad spectrum of RNA-seq workflows. Surpassing the expression analysis scope, our work also includes assessment of RNA variant-calling, RNA editing and RNA fusion detection techniques. Specifically, we examine both short- and long-read RNA-seq technologies, 39 analysis tools resulting in ~120 combinations, and ~490 analyses involving 15 samples with a variety of germline, cancer and stem cell data sets. We report the performance and propose a comprehensive RNA-seq analysis protocol, named RNACocktail, along with a computational pipeline achieving high accuracy. Validation on different samples reveals that our proposed protocol could help researchers extract more biologically relevant predictions by broad analysis of the transcriptome.RNA-seq is widely used for transcriptome analysis. Here, the authors analyse a wide spectrum of RNA-seq workflows and present a comprehensive analysis protocol named RNACocktail as well as a computational pipeline leveraging the widely used tools for accurate RNA-seq analysis.

Read more
September 22, 2019  |  

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Read more
September 22, 2019  |  

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

Read more
September 22, 2019  |  

Global identification of alternative splicing via comparative analysis of SMRT- and Illumina-based RNA-seq in strawberry.

Alternative splicing (AS) is a key post-transcriptional regulatory mechanism, yet little information is known about its roles in fruit crops. Here, AS was globally analyzed in the wild strawberry Fragaria vesca genome with RNA-seq data derived from different stages of fruit development. The AS landscape was characterized and compared between the single-molecule, real-time (SMRT) and Illumina RNA-seq platform. While SMRT has a lower sequencing depth, it identifies more genes undergoing AS (57.67% of detected multiexon genes) when it is compared with Illumina (33.48%), illustrating the efficacy of SMRT in AS identification. We investigated different modes of AS in the context of fruit development; the percentage of intron retention (IR) is markedly reduced whereas that of alternative acceptor sites (AA) is significantly increased post-fertilization when compared with pre-fertilization. When all the identified transcripts were combined, a total of 66.43% detected multiexon genes in strawberry undergo AS, some of which lead to a gain or loss of conserved domains in the gene products. The work demonstrates that SMRT sequencing is highly powerful in AS discovery and provides a rich data resource for later functional studies of different isoforms. Further, shifting AS modes may contribute to rapid changes of gene expression during fruit set.© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Read more
September 22, 2019  |  

Introduction to isoform sequencing using Pacific Biosciences technology (Iso-Seq)

Alternative RNA splicing is a known phenomenon, but we still do not have a complete catalog of isoforms that explain variability in the human transcriptome. We have made significant progress in developing methods to study variability of the transcriptome, but we are far away of having a complete picture of the transcriptome. The initial methods to study gene expression were based on cloning of cDNAs and Sanger sequencing. The strategy was labor-intensive and expensive. With the development of microarrays, different methods based on exon arrays and tiling arrays provided valuable information about RNA expression. However, the microarray presented significant limitations. Most of the limitations became apparent by 2005, but it was not until 2008 that an alternative method to study the transcriptome was developed. RNA Sequencing using next-generation sequencing (RNA-Seq) quickly became the technology of choice for gene expression profiling. Recently, the precision and sensitivity of RNA-Seq have come into question, especially for transcriptome reconstruction. This chapter will describe a relatively new method, “Isoform Sequencing (Iso-Seq). Iso-Seq was developed by Pacific Biosciences (PacBio), and it is capable of identifying new isoforms with extraordinary precision due to its long-read technology. The technique to create libraries is straightforward, and the PacBio RS II instrument generates the information in hours. The bioinformatics analysis is performed using the freely available SMRT® Portal software. The SMRT Portal is easy to use and capable of performing all the steps necessary to analyze the raw data and to generate high-quality full-length isoforms. For the universal acceptance of the Iso-Seq method, the capacity of the SMRT Cells needs to improve at least 10- to 100-fold to make the system affordable and attractive to users.

Read more
September 22, 2019  |  

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

The initiating nucleotide found at the 5′ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5’end of primary transcripts, but rather attempt to deplete non-targeted RNA.We developed a method, Cappable-seq, for directly enriching for the 5′ end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.Cappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.