skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: RNA Sequencing Data: Hitchhiker's Guide to Expression Analysis
Gene expression is the fundamental level at which the results of various genetic and regulatory programs are observable. The measurement of transcriptome-wide gene expression has convincingly switched from microarrays to sequencing in a matter of years. RNA sequencing (RNA-seq) provides a quantitative and open system for profiling transcriptional outcomes on a large scale and therefore facilitates a large diversity of applications, including basic science studies, but also agricultural or clinical situations. In the past 10 years or so, much has been learned about the characteristics of the RNA-seq data sets, as well as the performance of the myriad of methods developed. In this review, we give an overview of the developments in RNA-seq data analysis, including experimental design, with an explicit focus on the quantification of gene expression and statistical approachesfor differential expression. We also highlight emerging data types, such as single-cell RNA-seq and gene expression profiling using long-read technologies.  more » « less
Award ID(s):
1750472
PAR ID:
10132785
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Annual Review of Biomedical Data Science
Volume:
2
Issue:
1
ISSN:
2574-3414
Page Range / eLocation ID:
139 to 173
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; (, NAR Genomics and Bioinformatics)
    Abstract The ability to profile transcriptomes and characterize global gene expression changes has been greatly enabled by the development of RNA sequencing technologies (RNA-seq). However, the process of generating sequencing-compatible cDNA libraries from RNA samples can be time-consuming and expensive, especially for bacterial mRNAs which lack poly(A)-tails that are often used to streamline this process for eukaryotic samples. Compared to the increasing throughput and decreasing cost of sequencing, library preparation has had limited advances. Here, we describe bacterial-multiplexed-seq (BaM-seq), an approach that enables simple barcoding of many bacterial RNA samples that decreases the time and cost of library preparation. We also present targeted-bacterial-multiplexed-seq (TBaM-seq) that allows for differential expression analysis of specific gene panels with over 100-fold enrichment in read coverage. In addition, we introduce the concept of transcriptome redistribution based on TBaM-seq that dramatically reduces the required sequencing depth while still allowing for quantification of both highly and lowly abundant transcripts. These methods accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches. Together, use of these library preparation protocols allows for fast, affordable generation of sequencing libraries. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; ; et al (, Genome Biology)
    Abstract Background Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. Results We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. Conclusions AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species. 
    more » « less
  3. (, Genome biology)
    Background Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. Results We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. Conclusions AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species. 
    more » « less
  4. ; (, bioRxiv)
    Multi-modal single cell RNA assays capture RNA content as well as other data modalities, such as spatial cell position or the electrophysiological properties of cells. Compared to dedicated scRNA-seq assays however, they may unintentionally capture RNA from multiple adjacent cells, exhibit lower RNA sequencing depth compared to scRNA-seq, or lack genome-wide RNA measurements. We present scProjection, a method for mapping individual multi-modal RNA measurements to deeply sequenced scRNA-seq atlases to extract cell type-specific, single cell gene expression profiles. We demonstrate several use cases of scProjection, including the identification of spatial motifs from spatial transcriptome assays, distinguishing RNA contributions from neighboring cells in both spatial and multi-modal single cell assays, and imputing expression measurements of un-measured genes from gene markers. scProjection therefore combines the advantages of both multi-modal and scRNA-seq assays to yield precise multi-modal measurements of single cells. 
    more » « less
  5. ; ; (, Genome Research)
    Visualizing Ribo-seq and other sequencing data within genes of interest is a powerful approach to studying gene expression, but its application is limited by a lack of robust tools. Here, we introduce ggRibo, a user-friendly R package for visualizing individual gene expression, integrating Ribo-seq, RNA-seq, and other genome-wide datasets with flexible scaling options. ggRibo visualizes 3-nucleotide periodicity, a hallmark of translating ribosomes, within a gene-structure context, including introns and untranslated regions, enabling the study of novel ORFs, translation of different isoforms, and mechanisms of translational regulation. ggRibo can plot multiple Ribo-seq/RNA-seq datasets from different conditions for comparison. It also contains functions for plotting single-transcript view, reading-frame decomposition, and RNA-seq coverage alone. Importantly, ggRibo supports the visualization of other omics datasets that could also be presented with single-nucleotide resolution, such as RNA degradome, transcription start sites, translation initiation sites, and epitranscriptomic modifications. We demonstrate its utility with examples of upstream ORFs, downstream ORFs, nested ORFs, and differential isoform translation in humans,Arabidopsis, tomato, and rice. We also provide examples of multiomic comparisons that reveal insights that connect the transcriptome, translatome, and degradome. In summary, ggRibo is an advanced single-gene viewer that offers a valuable resource for studying gene expression regulation through its intuitive and flexible platform. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email