List of RNA-Seq bioinformatics tools

Quality control

• AfterQC AfterQC - Automatic Filtering, Trimming, Error Removing and Quality Control for fastq data.
• dupRadar dupRadar.^[7] An R package which provides functions for plotting and analyzing the duplication rates dependent on the expression levels.
• FastQC FastQC is a quality control tool for high-throughput sequence data (Babraham Institute) and is developed in Java. Import of data is possible from FastQ files, BAM or SAM format. This tool provides an overview to inform about problematic areas, summary graphs and tables to rapid assessment of data. Results are presented in HTML permanent reports. FastQC can be run as a stand-alone application or it can be integrated into a larger pipeline solution.
• fastqp fastqp. Simple FASTQ quality assessment using Python.
• Kraken kraken:^[8] A set of tools for quality control and analysis of high-throughput sequence data.
• HTSeq HTSeq.^[9] The Python script htseq-qa takes a file with sequencing reads (either raw or aligned reads) and produces a PDF file with useful plots to assess the technical quality of a run.
• mRIN mRIN^[10] - Assessing mRNA integrity directly from RNA-Seq data.
• MultiQC MultiQC^[11] - Aggregate and visualise results from numerous tools (FastQC, HTSeq, RSeQC, Tophat, STAR, others..) across all samples into a single report.
• NGSQC NGSQC: cross-platform quality analysis pipeline for deep sequencing data.
• NGS QC Toolkit NGS QC Toolkit A toolkit for the quality control (QC) of next generation sequencing (NGS) data. The toolkit comprises user-friendly stand alone tools for quality control of the sequence data generated using Illumina and Roche 454 platforms with detailed results in the form of tables and graphs, and filtering of high-quality sequence data. It also includes few other tools, which are helpful in NGS data quality control and analysis.
• PRINSEQ PRINSEQ is a tool that generates summary statistics of sequence and quality data and that is used to filter, reformat and trim next-generation sequence data. It is particular designed for 454/Roche data, but can also be used for other types of sequence.
• QC-Chain QC-Chain is a package of quality control tools for next generation sequencing (NGS) data, consisting of both raw reads quality evaluation and de novo contamination screening, which could identify all possible contamination sequences.
• QC3 QC3 a quality control tool designed for DNA sequencing data for raw data, alignment, and variant calling.
• qrqc qrqc. Quickly scans reads and gathers statistics on base and quality frequencies, read length, and frequent sequences. Produces graphical output of statistics for use in quality control pipelines, and an optional HTML quality report. S4 SequenceSummary objects allow specific tests and functionality to be written around the data collected.
• RNA-SeQC RNA-SeQC^[12] is a tool with application in experiment design, process optimization and quality control before computational analysis. Essentially, provides three types of quality control: read counts (such as duplicate reads, mapped reads and mapped unique reads, rRNA reads, transcript-annotated reads, strand specificity), coverage (like mean coverage, mean coefficient of variation, 5’/3’ coverage, gaps in coverage, GC bias) and expression correlation (the tool provides RPKM-based estimation of expression levels). RNA-SeQC is implemented in Java and is not required installation, however can be run using the GenePattern web interface. The input could be one or more BAM files. HTML reports are generated as output.
• RSeQC RSeQC^[13] analyzes diverse aspects of RNA-Seq experiments: sequence quality, sequencing depth, strand specificity, GC bias, read distribution over the genome structure and coverage uniformity. The input can be SAM, BAM, FASTA, BED files or Chromosome size file (two-column, plain text file). Visualization can be performed by genome browsers like UCSC, IGB and IGV. However, R scripts can also be used to visualization.
• SAMStat SAMStat ^[14] identifies problems and reports several statistics at different phases of the process. This tool evaluates unmapped, poorly and accurately mapped sequences independently to infer possible causes of poor mapping.
• SolexaQA SolexaQA calculates sequence quality statistics and creates visual representations of data quality for second-generation sequencing data. Originally developed for the Illumina system (historically known as “Solexa”), SolexaQA now also supports Ion Torrent and 454 data.
• Trim galore! Trim_galore is a wrapper script to automate quality and adapter trimming as well as quality control, with some added functionality to remove biased methylation positions for RRBS sequence files (for directional, non-directional (or paired-end) sequencing).

Improving the quality

Improvement of the RNA-Seq quality, correcting the bias is a complex subject.^[15][16] Each RNA-Seq protocol introduces specific type of bias, each step of the process (such as the sequencing technology used) is susceptible to generate some sort of noise or type of error. Furthermore, even the species under investigation and the biological context of the samples are able to influence the results and introduce some kind of bias. Many sources of bias were already reported – GC content and PCR enrichment,^[17][18] rRNA depletion,^[19] errors produced during sequencing,^[20] priming of reverse transcription caused by random hexamers.^[21]

Different tools were developed to attempt to solve each of the detected errors.

Short (unspliced) aligners

Short aligners are able to align continuous reads (not containing gaps result of splicing) to a genome of reference. Basically, there are two types: 1) based on the Burrows-Wheeler transform method such as Bowtie and BWA, and 2) based on Seed-extend methods, Needleman-Wunsch or Smith-Waterman algorithms. The first group (Bowtie and BWA) is many times faster, however some tools of the second group tend to be more sensitive, generating more correctly aligned reads. See a comparative study of short aligners - comparative study.

• BFAST BFAST aligns short reads to reference sequences and presents particular sensitivity towards errors, SNPs, insertions and deletions. BFAST works with the Smith-Waterman algorithm. See also seqanwers/BFAST.
• Bowtie Bowtie is a fast short aligner using an algorithm based on the Burrows-Wheeler transform and the FM-index. Bowtie tolerates a small number of mismatches.
• Burrows-Wheeler Aligner (BWA) BWA. BWA is a software package for mapping low-divergent sequences against a large reference genome, such as the human genome. It consists of three algorithms: BWA-backtrack, BWA-SW and BWA-MEM. The first algorithm is designed for Illumina sequence reads up to 100bp, while the rest two for longer sequences ranged from 70bp to 1Mbp. BWA-MEM and BWA-SW share similar features such as long-read support and split alignment, but BWA-MEM, which is the latest, is generally recommended for high-quality queries as it is faster and more accurate. BWA-MEM also has better performance than BWA-backtrack for 70-100bp Illumina reads.
• Short Oligonucleotide Analysis Package (SOAP) SOAP.
• GNUMAP GNUMAP performs alignment using a probabilistic Needleman-Wunsch algorithm. This tool is able to handle alignment in repetitive regions of a genome without losing information. The output of the program was developed to make possible easy visualization using available software.
• Maq Maq first aligns reads to reference sequences and after performs a consensus stage. On the first stage performs only ungapped alignment and tolerates up to 3 mismatches.
• Mosaik Mosaik. Mosaik is able to align reads containing short gaps using Smith-Waterman algorithm, ideal to overcome SNPs, insertions and deletions.
• NovoAlign (commercial) NovoAlign is a short aligner to the Illumina platform based on Needleman-Wunsch algorithm. Novoalign tolerates up to 8 mismatches per read, and up to 7bp of indels. It is able to deal with bisulphite data. Output in SAM format.
• PerM PerM is a software package which was designed to perform highly efficient genome scale alignments for hundreds of millions of short reads produced by the ABI SOLiD and Illumina sequencing platforms. PerM is capable of providing full sensitivity for alignments within 4 mismatches for 50bp SOLID reads and 9 mismatches for 100bp Illumina reads.
• RazerS RazerS.
• SEAL SEAL uses a MapReduce model to produce distributed computing on clusters of computers. Seal uses BWA to perform alignment and Picard MarkDuplicates to detection and duplicate read removal.
• segemehl segemehl.
• SeqMap SeqMap.
• SHRiMP SHRiMP employs two techniques to align short reads. Firstly, the q-gram filtering technique based on multiple seeds identifies candidate regions. Secondly, these regions are investigated in detail using Smith-Waterman algorithm.
• SMALT Smalt.
• Stampy Stampy combines the sensitivity of hash tables and the speed of BWA. Stampy is prepared to alignment of reads containing sequence variation like insertions and deletions. It is able to deal with reads up to 4500 bases and presents the output in SAM format.
• ZOOM (commercial) ZOOM is a short aligner of the Illumina/Solexa 1G platform. ZOOM uses extended spaced seeds methodology building hash tables for the reads, and tolerates mismatches and insertions and deletions.
• WHAM Wham. WHAM is a high-throughput sequence alignment tool developed at University of Wisconsin-Madison. It aligns short DNA sequences (reads) to the whole human genome at a rate of over 1500 million 60bit/s reads per hour, which is one to two orders of magnitudes faster than the leading state-of-the-art techniques.

Spliced aligners

Many reads span exon-exon junctions and can not be aligned directly by Short aligners, thus specific aligners were necessary - Spliced aligners. Some Spliced aligners employ Short aligners to align firstly unspliced/continuous reads (exon-first approach), and after follow a different strategy to align the rest containing spliced regions - normally the reads are split into smaller segments and mapped independently. See also.^[42][43]

Evaluation of alignment tools

• AlignerBoost AlignerBoost is a generalized software toolkit for boosting Next-Gen sequencing mapping precision using a Bayesian-based mapping quality framework.
• CADBURE CADBURE:Bioinformatics tool for evaluating aligner performance on your RNA-Seq dataset.
• QualiMap QualiMap: Evaluating next generation sequencing alignment data.
• RNAseqEVAL RNAseqEval A collection of tools for evaluating RNA seq mapping.
• Teaser Teaser: Individualized benchmarking and optimization of read mapping results for NGS data.

General tools

These tools perform normalization and calculate the abundance of each gene expressed in a sample.^[49] RPKM, FPKM and TPMs^[50] are some of the units employed to quantification of expression (RPKM-FPKM-TPMs video). Some software are also designed to study the variability of genetic expression between samples (differential expression). Quantitative and differential studies are largely determined by the quality of reads alignment and accuracy of isoforms reconstruction. Several studies are available comparing differential expression methods.^[51][52][53]

• ABSSeq ABSSeq -a new RNA-Seq analysis method based on modelling absolute expression differences.
• ALDEx2 ALDEx2 is a tool for comparative analysis of high-throughput sequencing data. ALDEx2 uses compositional data analysis and can be applied to RNAseq, 16S rRNA gene sequencing, metagenomic sequencing, and selective growth experiments.
• Alexa-Seq Alexa-Seq is a pipeline that makes possible to perform gene expression analysis, transcript specific expression analysis, exon junction expression and quantitative alternative analysis. Allows wide alternative expression visualization, statistics and graphs.
• ARH-seq – identification of differential splicing in RNA-seq data.
• ASC ASC.^[54]
• Ballgown Ballgown.
• BaySeq BaySeq is a Bioconductor package to identify differential expression using next-generation sequencing data, via empirical Bayesian methods. There is an option of using the "snow" package for parallelisation of computer data processing, recommended when dealing with large data sets.
• BBSeq BBSeq.
• BitSeq BitSeq. (Bayesian Inference of Transcripts from Sequencing Data) is an application for inferring expression levels of individual transcripts from sequencing (RNA-Seq) data and estimating differential expression (DE) between conditions.
• CEDER CEDER. Accurate detection of differentially expressed genes by combining significance of exons using RNA-Seq.
• CPTRA CPTRA. The CPTRA package is for analyzing transcriptome sequencing data from different sequencing platforms. It combines advantages of 454, Illumina GAII, or other platforms and can perform sequence tag alignment and annotation, expression quantification tasks.
• casper casper is a Bioconductor package to quantify expression at the isoform level. It combines using informative data summaries, flexible estimation of experimental biases and statistical precision considerations which (reportedly) provide substantial reductions in estimation error.
• Cufflinks/Cuffdiff Cufflinks is appropriate to measure global de novo transcript isoform expression. It performs assembly of transcripts, estimation of abundances and determines differential expression (Cuffdiff) and regulation in RNA-Seq samples.^[55]
• DESeq DESeq is a Bioconductor package to perform differential gene expression analysis based on negative binomial distribution.
• DEGSeq DEGSeq.
• Derfinder Derfinder Annotation-agnostic differential expression analysis of RNA-seq data at base-pair resolution via the DER Finder approach.
• DEXSeq DEXSeq is Bioconductor package that finds differential differential exon usage based on RNA-Seq exon counts between samples. DEXSeq employs negative binomial distribution, provides options to visualization and exploration of the results.
• DEXUS dexus is a Bioconductor package that identifies differentially expressed genes in RNA-Seq data under all possible study designs such as studies without replicates, without sample groups, and with unknown conditions.^[56] In contrast to other methods, DEXUS does not need replicates to detect differentially expressed transcripts, since the replicates (or conditions) are estimated by the EM method for each transcript.
• DGEclust dgeclust is a Python package for clustering expression data from RNA-seq, CAGE and other NGS assays using a Hierarchical Dirichlet Process Mixture Model. The estimated cluster configurations can be post-processed in order to identify differentially expressed genes and for generating gene- and sample-wise dendrograms and heatmaps.^[57]
• DiffSplice DiffSplice is a method for differential expression detection and visualization, not dependent on gene annotations. This method is supported on identification of alternative splicing modules (ASMs) that diverge in the different isoforms. A non-parametric test is applied to each ASM to identify significant differential transcription with a measured false discovery rate.
• EBSeq EBSeq is a Bioconductor package for identifying genes and isoforms differentially expressed (DE) across two or more biological conditions in an RNA-seq experiment. It also can be used to identify DE contigs after performing de novo transcriptome assembly. While performing DE analysis on isoforms or contigs, different isoform/contig groups have varying estimation uncertainties. EBSeq models the varying uncertainties using an empirical Bayes model with different priors.
• EdgeR EdgeR is a R package for analysis of differential expression of data from DNA sequencing methods, like RNA-Seq, SAGE or ChIP-Seq data. edgeR employs statistical methods supported on negative binomial distribution as a model for count variability.
• EdgeRun edgeRun: an R package for sensitive, functionally relevant differential expression discovery using an unconditional exact test.
• EQP EQP - The exon quantification pipeline (EQP): a comprehensive approach to the quantification of gene, exon and junction expression from RNA-seq data.
• ESAT ESAT The End Sequence Analysis Toolkit (ESAT) is specially designing to be applied for quantification of annotation of specialized RNA-Seq gene libraries that target the 5' or 3' ends of transcripts.
• eXpress eXpress performance includes transcript-level RNA-Seq quantification, allele-specific and haplotype analysis and can estimate transcript abundances of the multiple isoforms present in a gene. Although could be coupled directly with aligners (like Bowtie), eXpress can also be used with de novo assemblers and thus is not needed a reference genome to perform alignment. It runs on Linux, Mac and Windows.
• ERANGE ERANGE performs alignment, normalization and quantification of expressed genes.
• featureCounts featureCounts an efficient general-purpose read quantifier. It is part of the SourceForge Subread package and Bioconductor Rsubread package.
• FDM FDM.
• FineSplice FineSplice - Enhanced splice junction detection and estimation from RNA-Seq data.
• GFOLD GFOLD:^[58] Generalized fold change for ranking differentially expressed genes from RNA-seq data.
• globalSeq globalSeq.^[59] Global test for counts: testing for association between RNA-Seq and high-dimensional data.
• GPSeq GPSeq This is a software tool to analyze RNA-seq data to estimate gene and exon expression, identify differentially expressed genes, and differentially spliced exons.
• IsoDOT – Differential RNA-isoform Expression.
• Limma Limma. Limma powers differential expression analyses for RNA-sequencing and microarray studies.
• LPEseq LPEseq – accurately test differential expression with a limited number of replicates.
• Kallisto Kallisto. "Kallisto is a program for quantifying abundances of transcripts from RNA-Seq data, or more generally of target sequences using high-throughput sequencing reads. It is based on the novel idea of pseudoalignment for rapidly determining the compatibility of reads with targets, without the need for alignment. On benchmarks with standard RNA-Seq data, kallisto can quantify 30 million human reads in less than 3 minutes on a Mac desktop computer using only the read sequences and a transcriptome index that itself takes less than 10 minutes to build."
• MATS MATS. Multivariate Analysis of Transcript Splicing (MATS).
• MetaDiff MetaDiff. Differential isoform expression analysis using random-effects meta-regression.
• metaseqR metaseqR is a Bioconductor package that detects differentially expressed genes from RNA-Seq data by combining six statistical algorithms using weights estimated from their performance with simulated data estimated from real data, either public or user-based. In this way, metaseqR optimizes the tradeoff between precision and sensitivity.^[60] In addition, metaseqR creates a detailed and interactive report with a variety of diagnostic and exploration plots and auto-generated text.
• MMSEQ MMSEQ is a pipeline for estimating isoform expression and allelic imbalance in diploid organisms based on RNA-Seq. The pipeline employs tools like Bowtie, TopHat, ArrayExpressHTS and SAMtools. Also, edgeR or DESeq to perform differential expression.
• MultiDE multiDE
• Myrna Myrna is a pipeline tool that runs in a cloud environment (Elastic MapReduce) or in a unique computer for estimating differential gene expression in RNA-Seq datasets. Bowtie is employed for short read alignment and R algorithms for interval calculations, normalization, and statistical processing.
• NEUMA NEUMA is a tool to estimate RNA abundances using length normalization, based on uniquely aligned reads and mRNA isoform models. NEUMA uses known transcriptome data available in databases like RefSeq.
• NOISeq NOISeq. NOISeq is a non-parametric approach for the identification of differentially expressed genes from count data or previously normalized count data. NOISeq empirically models the noise distribution of count changes by contrasting fold-change differences (M) and absolute expression differences (D) for all the features in samples within the same condition.
• NPEBseq NPEBseq is a nonparametric empirical bayesian- based method for differential expression analysis.
• NSMAP NSMAP allows inference of isoforms as well estimation of expression levels, without annotated information. The exons are aligned and splice junctions are identified using TopHat. All the possible isoforms are computed by combination of the detected exons.
• NURD NURD an implementation of a new method to estimate isoform expression from non-uniform RNA-seq data.
• PANDORA PANDORA. An R package for the analysis and result reporting of RNA-Seq data by combining multiple statistical algorithms.
• PennSeq PennSeq PennSeq: accurate isoform-specific gene expression quantification in RNA-Seq by modeling non-uniform read distribution.
• Quark Quark - Quark enables semi-reference-based compression of RNA-seq data.
• QuasR QuasR - Quantify and Annotate Short Reads in R.
• RapMap RapMap. A Rapid, Sensitive and Accurate Tool for Mapping RNA-seq Reads to Transcriptomes.
• RNAeXpress RNAeXpress Can be run with Java GUI or command line on Mac, Windows and Linux. Can be configured to perform read counting, feature detection or GTF comparison on mapped rnaseq data.
• Rcount Rcount. Rcount: simple and flexible RNA-Seq read counting.
• rDiff rDiff is a tool that can detect differential RNA processing (e.g. alternative splicing, polyadenylation or ribosome occupancy).
• RNASeqPower RNASeqPower. Calculating samples Size estimates for RNA Seq studies. R package version .
• RNA-Skim RNA-Skim. RNA-Skim: a rapid method for RNA-Seq quantification at transcript-level.
• rSeq rSeq. rSeq is a set of tools for RNA-Seq data analysis. It consists of programs that deal with many aspects of RNA-Seq data analysis, such as read quality assessment, reference sequence generation, sequence mapping, gene and isoform expressions (RPKMs) estimation, etc.
• RSEM RSEM.
• rQuant rQuant is a web service (Galaxy (computational biology) installation) that determines abundances of transcripts per gene locus, based on quadratic programming. rQuant is able to evaluate biases introduced by experimental conditions. A combination of tools is employed: PALMapper (reads alignment), mTiM and mGene (inference of new transcripts).
• Salmon Salmon is an software tool for computing transcript abundance from RNA-seq data using either an alignment-free (based directly on the raw reads) or an alignment-based (based on pre-computed alignments) approach. It uses an online stochastic optimization approach to maximize the likelihood of the transcript abundances under the observed data. The software itself is capable of making use of many threads to produce accurate quantification estimates quickly. It is part of the Sailfish suite of software, and is the successor to the Sailfish tool.
• SAJR SAJR is a java-written read counter and R-package for differential splicing analysis. It uses junction reads to estimate exon exclusion and reads mapped within exon to estimate its inclusion. SAJR models it by GLM with quasibinomial distribution and uses log likelihood test to assess significance.
• Scotty Scotty Performs power analysis to estimate the number of replicates and depth of sequencing required to call differential expression.
• Seal Seal Ultrafast, alignment-free algorithm to quantify sequence expression by matching kmers between raw reads and a reference transcriptome. Handles paired reads and alternate isoforms, and uses little memory. Accepts all common read formats, and outputs read counts, coverage, and FPKM values per reference sequence. Open-source, written in pure Java; supports all platforms with no recompilation and no other dependencies. Distributed with BBMap. (Seal - Sequence Expression AnaLyzer - is unrelated to the SEAL distributed short-read aligner.)
• semisup semisup.^[61] Semi-supervised mixture model: detecting SNPs with interactive effects on a quantitative trait
• Sleuth Sleuth is a program for analysis of RNA-Seq experiments for which transcript abundances have been quantified with kallisto.
• SplicingCompass SplicingCompass. SplicingCompass: differential splicing detection using RNA-Seq data.
• sSeq sSeq. The purpose of this R package is to discover the genes that are differentially expressed between two conditions in RNA-seq experiments.
• StringTie StringTie is a fast and highly efficient assembler of RNA-Seq alignments into potential transcripts. It uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus. It was designed as a successor to Cufflinks (its developers include some of the Cufflinks developers) and has many of the same features, but runs far faster and in far less memory.
• TIGAR TIGAR Transcript isoform abundance estimation method with gapped alignment of RNA-Seq data by variational Bayesian inference.
• TimeSeq TimeSeq - Detecting Differentially Expressed Genes in Time Course RNA-Seq Data.
• WemIQ WemIQis a software tool to quantify isoform expression and exon splicing ratios from RNA-seq data accurately and robustly.

Evaluation of quantification and differential expression

• CompcodeR CompcodeR— RNAseq data simulation, differential expression analysis and performance comparison of differential expression methods.
• DEAR-O DEAR-O : Differential Expression Analysis based on RNA-seq data – Online.
• PROPER PROPER: comprehensive power evaluation for differential expression using RNA-seq.
• RNAontheBENCH RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods.
• rnaseqcomp rnaseqcomp. Several quantitative and visualized benchmarks for RNA-seq quantification pipelines. Two-condition quantifications for genes, transcripts, junctions or exons by each pipeline with nessasery meta information should be organizd into numeric matrices in order to proceed the evaluation.
• http://www.clipz.unibas.ch/benchmarking. Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data.

Multi-tool solutions

• DEB DEB is a web-interface/pipeline that permits to compare results of significantly expressed genes from different tools. Currently are available three algorithms: edgeR, DESeq and bayseq.
• SARTools SARTools: A DESeq2- and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data.

Commercial solutions

• ActiveSite by Cofactor Genomics ActiveSite
• Avadis NGS (currently Strand NGS) Strand NGS.
• BaseSpace by Illumina BaseSpace
• Biowardrobe Biowardrobe an integrated platform for analysis of epigenomics and transcriptomics data.
• CLC Genomics Workbench CLC Genomics Workbench
• DNASTAR DNASTAR
• ERGO ERGO
• Genedata
• GeneSpring GX GeneSpring GX
• Genevestigator by Nebion Genevestigator A userfriendly platform for transcriptomic meta-analysis of public data.
• geospiza geospiza
• Golden Helix Golden Helix
• Maverix Biomics Maverix Biomics
• NextGENe NextGENe
• OmicsOffice OmicsOffice
• Qlucore. Easy to use for analysis and visualization. One button import of BAM files.

Open (free) source solutions

• ArrayExpressHTS ArrayExpressHTS (and ebi_ArrayExpressHTS) is a BioConductor package that allows preprocessing, quality assessment and estimation of expression of RNA-Seq datasets. It can be run remotely at the European Bioinformatics Institute cloud or locally. The package makes use of several tools: ShortRead (quality control), Bowtie, TopHat or BWA (alignment to a reference genome), SAMtools format, Cufflinks or MMSEQ (expression estimation).
• BioQueue BioQueue is a web-based queue engine designed preferentially to improve the efficiency and robustness of job execution in bioinformatics research by estimating the system resources required by a certain job. At the same time, BioQueue also aims to promote the accessibility and reproducibility of data analysis in biomedical research. Implemented by Python 2.7, BioQueue can work in both POSIX compatible systems (Linux, Solaris, OS X, etc.) and Windows. See also.^[62]
• BioWardrobe BioWardrobe is an integrated package that for analysis of ChIP-Seq and RNA-Seq datasets using a web-based user-friendly GUI. For RNA-Seq Biowardrobe performs mapping, quality control, RPKM estimation and differential expression analysis between samples (groups of samples). Results of differential expression analysis can be integrated with ChIP-Seq data to build average tag density profiles and heat maps. The package makes use of several tools open source tools including STAR and DESeq. See also.^[63]
• Chipster Chipster. Chipster is a user-friendly analysis software for high-throughput data. It contains over 350 analysis tools for next generation sequencing (NGS), microarray, proteomics and sequence data. Users can save and share automatic analysis workflows, and visualize data interactively using a built-in genome browser and many other visualizations.
• DEWE DEWE. DEWE (Differential Expression Workflow Executor) is an open source desktop application that provides a user-friendly GUI for easily executing Differential Expression analyses in RNA-Seq data. Currently, DEWE provides two differential expression analysis workflows: HISAT2, StringTie and Ballgown and Bowtie2, StringTie and R libraries (Ballgown and edgeR). It runs in Linux, Windows and Mac OS X.
• easyRNASeq easyRNASeq. Calculates the coverage of high-throughput short-reads against a genome of reference and summarizes it per feature of interest (e.g. exon, gene, transcript). The data can be normalized as 'RPKM' or by the 'DESeq' or 'edgeR' package.
• ExpressionPlot ExpressionPlot.
• FX FX. FX is a user-Frendly RNA-Seq gene eXpression analysis tool, empowered by the concept of cloud-computing. With FX, you can simply upload your RNA-Seq raw FASTQ data on the cloud, and let the computing infra to do the heavy analysis.
• Galaxy: Galaxy is a general purpose workbench platform for computational biology. There are several publicly accessible Galaxy servers that support RNA-Seq tools and workflows, including NBIC's Andromeda, the CBIIT-Giga server, the Galaxy Project's public server, the GeneNetwork Galaxy server, the University of Oslo's Genomic Hyperbrowser, URGI's server (which supports S-MART), and many others.
• GENE-Counter GENE-Counter is a Perl pipeline for RNA-Seq differential gene expression analyses. Gene-counter performs alignments with CASHX, Bowtie, BWA or other SAM output aligner. Differential gene expression is run with three optional packages (NBPSeq, edgeR and DESeq) using negative binomial distribution methods. Results are stored in a MySQL database to make possible additional analyses.
• GenePattern GenePattern offers integrated solutions to RNA-Seq analysis (Broad Institute).
• GeneProf GeneProf: Freely accessible, easy to use analysis pipelines for RNA-seq and ChIP-seq experiments.
• GREIN GREIN is an interactive web platform for re-processing and re-analyzing GEO RNA-seq data. GREIN is powered by the back-end computational pipeline for uniform processing of RNA-seq data and the large number (>5,800) of already processed data sets. The front-end user friendly interfaces provide a wealth of user-analytics options including sub-setting and downloading processed data, interactive visualization, statistical power analyses, construction of differential gene expression signatures and their comprehensive functional characterization, connectivity analysis with LINCS L1000 data, etc.
• GT-FAR GT-FAR is an RNA seq pipeline that performs RNA-seq QC, alignment, reference free quantification, and splice variant calling. It filters, trims, and sequentially aligns reads to gene models and predicts and validates new splice junctions after which it quantifies expression for each gene, exon, and known/novel splice junction, and Variant Calling.
• MultiExperiment Viewer (MeV) MeV is suitable to perform analysis, data mining and visualization of large-scale genomic data. The MeV modules include a variety of algorithms to execute tasks like Clustering and Classification, Student's t-test, Gene Set Enrichment Analysis or Significance Analysis. MeV runs on Java. See also seqanswers/MeV.
• NGSUtils NGSUtils. NGSUtils is a suite of software tools for working with next-generation sequencing datasets.
• Rail-RNA Rail-Rna. Scalable analysis of RNA-seq splicing and coverage.
• RAP - RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.
• RSEQtools RSEQtools. "RSEQtools consists of a set of modules that perform common tasks such as calculating gene expression values, generating signal tracks of mapped reads, and segmenting that signal into actively transcribed regions. In addition to the anonymization afforded by this format it also facilitates the decoupling of the alignment of reads from downstream analyses."
• RobiNA RobiNA provides a user graphical interface to deal with R/BioConductor packages. RobiNA provides a package that automatically installs all required external tools (R/Bioconductor frameworks and Bowtie). This tool offers a diversity of quality control methods and the possibility to produce many tables and plots supplying detailed results for differential expression. Furthermore, the results can be visualized and manipulated with MapMan and PageMan. RobiNA runs on Java version 6.
• RseqFlow RseqFlow is an RNA-Seq analysis pipeline which offers an express implementation of analysis steps for RNA sequencing datasets. It can perform pre and post mapping quality control (QC) for sequencing data, calculate expression levels for uniquely mapped reads, identify differentially expressed genes, and convert file formats for ease of visualization.
• S-MART S-MART handles mapped RNA-Seq data, and performs essentially data manipulation (selection/exclusion of reads, clustering and differential expression analysis) and visualization (read information, distribution, comparison with epigenomic ChIP-Seq data). It can be run on any laptop by a person without computer background. A friendly graphical user interface makes easy the operation of the tools.
• Taverna Taverna. Taverna is an open source and domain-independent Workflow Management System – a suite of tools used to design and execute scientific workflows and aid in silico experimentation.
• TCW TCW. TCW is a Transcriptome Computational Workbench.
• TRAPLINE TRAPLINE: a standardized and automated pipeline for RNA sequencing data analysis, evaluation and annotation.
• ViennaNGS ViennaNGS. A toolbox for building efficient next- generation sequencing analysis pipelines.
• wapRNA wapRNA. This is a free web-based application for the processing of high-throughput RNA-Seq data (wapRNA) from next generation sequencing (NGS) platforms, such as Genome Analyzer of Illumina Inc. (Solexa) and SOLiD of Applied Biosystems (SOLiD). wapRNA provides an integrated tool for RNA sequence, refers to the use of High-throughput sequencing technologies to sequence cDNAs in order to get information about a sample's RNA content.

General tools

• Alternative Splicing Analysis Tool Package(ASATP) ASATP. Alternative splicing analysis tool package (ASATP) includes a series of toolkits to analyze alternative splicing events, which could be used to detect and visualized alternative splicing events, check ORF changes, assess regulations of alternative splicing and do statistical analysis.
• Asprofile asprofile. ASprofile is a suite of programs for extracting, quantifying and comparing alternative splicing (AS) events from RNA-seq data.
• AStalavista AStalavista. The AStalavista web server extracts and displays alternative splicing (AS) events from a given genomic annotation of exon-intron gene coordinates. By comparing all given transcripts, AStalavista detects the variations in their splicing structure and identify all AS events (like exon skipping, alternate donor, etc.) by assigning to each of them an AS code.
• CLASS2 Class: accurate and efficient splice variant annotation from RNA-seq reads.
• Cufflinks/Cuffdiff Cufflinks.
• DEXseq DEXseq. Inference of differential exon usage in RNA-Seq.
• Diceseq Diceseq - Statistical modeling of isoform splicing dynamics from RNA-seq time series data.
• EBChangepoint EBChangepoint An empirical Bayes change-point model for identifying 3′ and 5′ alternative splicing by RNA-Seq.
• Eoulsan Eoulsan A versatile framework dedicated to high throughput sequencing data analysis. Allows automated analysis (mapping, counting and differencial analysis with DESeq2).
• GESS GESS – for de novo detection of exon-skipping event sites from raw RNA-seq reads.
• LeafCutter LeaffCutter, a suite of novel methods that allow identification and quantication of novel and existing alternative splicing events by focusing on intron excisions.
• LEMONS ^[64] A Tool for the Identification of Splice Junctions in Transcriptomes of Organisms Lacking Reference Genomes.
• MAJIQ MAJIQ. Modeling Alternative Junction Inclusion Quantification.
• MATS MATS. Multivariate Analysis of Transcript Splicing (MATS).
• MISO MISO quantifies the expression level of splice variants from RNA-Seq data and is able to recognize differentially regulated exons/isoforms across different samples. MISO uses a probabilistic method (Bayesian inference) to calculate the probability of the reads origin.
• Rail-RNA Rail-Rna. Scalable analysis of RNA-seq splicing and coverage.
• RPASuite ^[65] RPASuite. RPASuite (RNA Processing Analysis Suite) is a computational pipeline to identify differentially and coherently processed transcripts using RNA-seq data obtained from multiple tissue or cell lines.
• RSVP RSVP. RSVP is a software package for prediction of alternative isoforms of protein-coding genes, based on both genomic DNA evidence and aligned RNA-seq reads. The method is based on the use of ORF graphs, which are more general than the splice graphs used in traditional transcript assembly.
• SAJR SAJR calculates the number of the reads that confirms segment (part of gene between two nearest splice sites) inclusion or exclusion and then model these counts by GLM with quasibinomial distribution to account for biological variability.
• SGSeq SGSeq A R package to de novo prediction of splicing events.
• SplAdder SplAdder Identification, quantification and testing of alternative splicing events from RNA-Seq data.
• SpliceGrapher Prediction of novel alternative splicing events from RNA-Seq data.^[66]
• SpliceJumper SpliceJumper: a classification-based approach for calling splicing junctions from RNA-seq data.
• SplicePie SplicePieis a pipeline to analyze non-sequential and multi-step splicing. SplicePie contains three major analysis steps: analyzing the order of splicing per sample, looking for recursive splicing events per sample and summarizing predicted recursive splicing events for all analyzed sample (it is recommended to use more samples for higher reliability). The first two steps are performed individually on each sample and the last step looks at the overlap in all samples. However, the analysis can be run on one sample as well.
• SplicePlot SplicePlot. SplicePlot is a tool for visualizing alternative splicing and the effects of splicing quantitative trait loci (sQTLs) from RNA-seq data. It provides a simple command line interface for drawing sashimi plots, hive plots, and structure plots of alternative splicing events from .bam, .gtf, and .vcf files.
• SpliceR SpliceR. An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data.
• SpliceSEQ SpliceSeq. SpliceViewer is a Java application that allows researchers to investigate alternative mRNA splicing patterns in data from high-throughput mRNA sequencing studies. Sequence reads are mapped to splice graphs that unambiguously quantify the inclusion level of each exon and splice junction. The graphs are then traversed to predict the protein isoforms that are likely to result from the observed exon and splice junction reads. UniProt annotations are mapped to each protein isoform to identify potential functional impacts of alternative splicing.
• SpliceTrap^[67] SpliceTrap is a statistical tool for the quantification of exon inclusion ratios from RNA-seq data.
• Splicing Express – a software suite for alternative splicing analysis using next-generation sequencing data.
• SUPPA Suppa. This tool generates different Alternative Splicing (AS) events and calculates the PSI ("Percentage Spliced In") value for each event exploiting the fast quantification of transcript abundances from multiple samples.
• SwitchSeq SwitchSeq identifies extreme changes in splicing (switch events).
• Portcullis Portcullis - identification of genuine splice junctions.
• TrueSight TrueSight A Self-training Algorithm for Splice Junction Detection using RNA-seq.
• Vast-tools vast-toolsA toolset for profiling alternative splicing events in RNA-Seq data.

Intron retention analysis

• IRcall / IRclassifier IRcall / IRclassifier. IRcall is a computational tool for IR event detection from RNA-Seq data. IRclassifier is a supervised machine learning-based approach for IR event detection from RNA-Seq data.

Differential isoform/transcript usage

• IsoformSwitchAnalyzeR IsoformSwitchAnalyzeR. IsoformSwitchAnalyzeR is an R package that enables statistical identification of isoform switches with predicted functional consequences where the consequences of interest can be chosen from a long list but includes gain/loss of protein domains, signal peptides changes in NMD sensitivity.^[68] IsoformSwitchAnalyzeR is made for post analysis of data from any full length isoform/transcript quantification tool but directly support Cufflinks/Cuffdiff, RSEM Kallisto and Salmon.
• DRIMSeq DRIMSeq. An R package that utilizes generalized linear modeling (GLM) to identify isoform switches from estimated isoform count data.^[69]
• BayesDRIMSeq BayesDRIMSeq. An R package conatining a Bayesian implementation of DRIMSeq.^[70]
• Cufflinks/Cuffdiff Cufflinks/Cuffdiff. Full length isoform/transcript quantification and differential analysis tool which amongst other test for changes in usage for isoform belonging to the same primary transcript (sharing a TSS) via a one-sided t-test based on the asymptotic of the Jensen-Shannon metric.^[71]
• rSeqNP rSeqNP. An R package that implements a non-parametric approach to test for differential expression and splicing from RNA-Seq data.^[72]
• Isolator Isolator. Full length isoform/transcript quantification and differential analysis tool which analyses all samples in an experiment in unison using a simple Bayesian hierarchical model. Can identify differential isoform usage by testing for probability of monotonic splicing.^[73]

Integrated Packages

• Monocle Monocle: Differential expression and time-series analysis for single-cell RNA-Seq and qPCR experiments.
• SCell SCell – integrated analysis of single-cell RNA-seq data.
• Sincell Sincell: an R/Bioconductor package for statistical assessment of cell-state hierarchies from single-cell RNA-seq.
• SINCERA SINCERA.^[87] A Pipeline for Single-Cell RNA-Seq Profiling Analysis.

Quality Control and Gene Filtering

• Celloline Celloline A pipeline for mapping and quality assessment single cell RNA-seq data.
• OEFinder OEFinder. A user interface to identify and visualize ordering effects in single-cell RNA-seq data.
• SinQC SinQC A Method and Tool to Control Single-cell RNA-seq Data Quality.

Normalization

• BASICS BASICS. Understanding changes in gene expression at the single-cell level.
• GRM GRM - Normalization and noise reduction for single cell RNA-seq experiments.

Dimension Reduction

• ZIFA ZIFA:^[88] Dimensionality reduction for zero-inflated single-cell gene expression analysis.

Differential Expression

• BPSC BPSC An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq.
• MAST MAST : a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data.
• SCDE SCDE. Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis.

Visualization

• eXpose

Genome-guided assemblers

• Bayesembler Baysembler Bayesian transcriptome assembly.
• CIDANE Cidane a comprehensive isoform discovery and abundance estimation.
• CLASS CLASS. CLASS is a program for assembling transcripts from RNA-seq reads aligned to a genome. CLASS produces a set of transcripts in three stages. Stage 1 uses linear programming to determine a set of exons for each gene. Stage 2 builds a splice graph representation of a gene, by connecting the exons (vertices) via introns (edges) extracted from spliced read alignments. Stage 3 selects a subset of the candidate transcripts encoded in the graph that can explain all the reads, using either a parsimonius (SET_COVER) or a dynamic programming optimization approach. This stage takes into account constraints derived from mate pairs and spliced alignments and, optionally, knowledge about gene structure extracted from known annotation or alignments of cDNA sequences.
• Cufflinks Cufflinks. Cufflinks assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples. It accepts aligned RNA-Seq reads and assembles the alignments into a parsimonious set of transcripts. Cufflinks then estimates the relative abundances of these transcripts based on how many reads support each one, taking into account biases in library preparation protocols.
• iReckon iReckon. iReckon is an algorithm for the simultaneous isoform reconstruction and abundance estimation. In addition to modelling novel isoforms, multi-mapped reads and read duplicates, this method takes into account the possible presence of unspliced pre-mRNA and intron retention. iReckon only requires a set of transcription start and end sites, but can use known full isoforms to improve sensitivity. Starting from the set of nearly all possible isoforms, iReckon uses a regularized EM algorithm to determine those actually present in the sequenced sample, together with their abundances. iReckon is multi-threaded to increase efficiency in all its time consuming steps.
• IsoInfer IsoInfer. IsoInfer is a C/C++ program to infer isoforms based on short RNA-Seq (single-end and paired-end) reads, exon-intron boundary and TSS/PAS information.
• IsoLasso IsoLasso. IsoLasso is an algorithm to assemble transcripts and estimate their expression levels from RNA-Seq reads.
• Flipflop flipflop. FlipFlop implements a fast method for de novo transcript discovery and abundance estimation from RNA-Seq data. It differs from Cufflinks by simultaneously performing the identification and quantitation tasks using a convex penalized maximum likelihood approach, which leads to improved precision/recall.
• GIIRA GIIRA. GIIRA is a gene prediction method that identifies potential coding regions exclusively based on the mapping of reads from an RNA-Seq experiment. It was foremost designed for prokaryotic gene prediction and is able to resolve genes within the expressed region of an operon. However, it is also applicable to eukaryotes and predicts exon intron structures as well as alternative isoforms.
• MITIE MITIE. Simultaneous RNA-Seq-based Transcript Identification and Quantification in Multiple Samples.
• RNAeXpress RNAeXpress. RNA-eXpress was designed as a user friendly solution to extract and annotate biologically important transcripts from next generation RNA sequencing data. This approach complements existing gene annotation databases by ensuring all transcripts present in the sample are considered for further analysis.
• Scripture Scripture. Scripture is a method for transcriptome reconstruction that relies solely on RNA-Seq reads and an assembled genome to build a transcriptome ab initio. The statistical methods to estimate read coverage significance are also applicable to other sequencing data. Scripture also has modules for ChIP-Seq peak calling.
• SLIDE SLIDE - Sparse Linear modeling of RNA-Seq data for Isoform Discovery and abundance Estimation.
• Strawberry Strawberry- A program for fast and accurate genome-guided transcripts reconstruction and quantification from paired-end RNA-seq.
• StringTie StringTie. StringTie is a fast and highly efficient assembler of RNA-Seq alignments into potential transcripts. It uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus. Its input can include not only the alignments of raw reads used by other transcript assemblers, but also alignments longer sequences that have been assembled from those reads.To identify differentially expressed genes between experiments, StringTie's output can be processed either by the Cuffdiff or Ballgown programs.
• TransComb TransComb a genome-guided transcriptome assembly via combing junctions in splicing graphs.
• Traph Traph A tool for transcript identification and quantification with RNA-Seq.
• Tiling Assembly for Annotation-independent Novel Gene Discovery.

Genome-independent (de novo) assemblers

• Bridger Bridger^[91] was developed at Shandong University, takes advantage of techniques employed in Cufflinks to overcome limitations of the existing de novo assemblers.
• CLC CLC de novo assembly algorithm of CLC Genomics Workbench.
• KISSPLICE KISSPLICE is a software that enables to analyse RNA-seq data with or without a reference genome. It is an exact local transcriptome assembler that allows to identify SNPs, indels and alternative splicing events. It can deal with an arbitrary number of biological conditions, and will quantify each variant in each condition.
• Oases Oases. De novo transcriptome assembler for very short reads.
• rnaSPAdes rnaSPAdes
• Rnnotator Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads.
• SAT-Assembler SAT-Assembler
• SOAPdenovo-Trans SOAPdenovo-trans.
• Scaffolding Translation Mapping STM.
• Trans-ABySS Trans-AByss.
• T-IDBA T-IDBA.
• Trinity Trinity was developed at the Broad Institute and the Hebrew University of Jerusalem, represents a novel method for the efficient and robust de novo reconstruction of transcriptomes from RNA-seq data. Trinity combines three independent software modules: Inchworm, Chrysalis, and Butterfly, applied sequentially to process large volumes of RNA-seq reads.
• Velvet Velvet (algorithm).^[92] Velvet(EMBL-EBI).

Assembly evaluation tools

• Busco Busco provides quantitative measures for the assessment of genome assembly, gene set, and transcriptome completeness, based on evolutionarily-informed expectations of gene content from near-universal single-copy orthologs selected from OrthoDB tool.
• Detonate Detonate. DETONATE (DE novo TranscriptOme rNa-seq Assembly with or without the Truth Evaluation) consists of two component packages, RSEM-EVAL and REF-EVAL. Both packages are mainly intended to be used to evaluate de novo transcriptome assemblies, although REF-EVAL can be used to compare sets of any kinds of genomic sequences.
• rnaQUAST rnaquast: Quality Assessment Tool for Transcriptome Assemblies.
• TransRate TransRate. Transrate is software for de-novo transcriptome assembly quality analysis. It examines your assembly in detail and compares it to experimental evidence such as the sequencing reads, reporting quality scores for contigs and assemblies. This allows you to choose between assemblers and parameters, filter out the bad contigs from an assembly, and help decide when to stop trying to improve the assembly.

Human related

• Brain RNA-Seq Brain RNA-Seq.^[110] An RNA-Seq transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.
• FusionCancer ^[111] FusionCancer: a database of cancer fusion genes derived from RNA-seq data.
• Hipposeq Hipposeq a comprehensive RNA-seq database of gene expression in hippocampal principal neurons.
• Mitranscriptome Mitranscriptome is a systematic list of long poly-adenylated Human RNA transcripts based on RNA-Seq data from more than 6,500 samples associated with a variety of cancer and tissue types. The database contains detailed gene expression analysis of over 91,000 genes, most are uncharacterized long RNAs.
• RNA-Seq Atlas^[112]RNA-Seq Atlas a reference database for gene expression profiling in normal tissue by next-generation sequencing.
• SRA SRA. The Sequence Read Archive (SRA) stores raw sequence data from "next-generation" sequencing technologies including 454, IonTorrent, Illumina, SOLiD, Helicos and Complete Genomics. In addition to raw sequence data, SRA now stores alignment information in the form of read placements on a reference sequence.
• DASHR DASHR. A database of human small RNA genes and mature products derived from small RNA-seq data.

Single species' RNA-Seq databases

• Aedes-albopictus Aedes albopictus Aedes albopictus database.
• Arabidopsis thaliana TraVa the database of gene expression profiles in Arabidopsis thaliana based on RNA-seq analysis.
• Barley morexGenes- Barley RNA-seq Database.
• Chickpea Chickpea transcriptome database (CTDB) has been developed with the view to provide most comprehensive information about the chickpea transcriptome, the most relevant part of the genome".
• Chilo suppressalis ChiloDB : a genomic and transcriptome database for an important rice insect pest Chilo suppressalis.
• Echinoderm EchinoDB – a repository of orthologous transcripts from echinoderms.
• Equine transcriptome (University of California, Davis). The data can be found at: https://github.com/dib-lab/horse_trans.
• Escherichia coli Ecomics – an omics normalized database for Escherichia coli.
• Fish Phylofish.
• Ginger Ginger Ginger transcriptome database.
• Lygodium japonicum Lygodium japonicum Transcriptome Database.
• Mammals Mammalian Transcriptomic Database.
• Oyster (Pacific) GigaTon: an extensive publicly searchable database providing a new reference transcriptome in the pacific oyster Crassostrea gigas.
• Mouse and Human PanglaoDB: A gene expression database for exploration and meta-analysis of single cell sequencing data.
• Mangrove Mangrove Transcriptome Database.
• Krill (Antarctic) KrillDB: a de novo Transcriptome Database for the Antarctic Krill.
• Mouse RNASeqMetaDB: a database and web server for navigating metadata of publicly available mouse RNA-Seq data sets.
• Rubus Rubus GDR RefTrans V1 - GDR Rubus RefTrans combines published RNA-Seq and EST data sets to create a reference transcriptome (RefTrans) for rubus and provides putative gene function identified by homology to known proteins.
• Sorghum MOROKOSHI Sorghum transcriptome database. RIKEN full-length cDNA clone and RNA-Seq data in Sorghum bicolor.
• S. purpuratus S. purpuratus Developmental Transcriptomes of S. purpuratus
• S. cerevisiae YeastMine transcriptome database.
• Wheat WheatExp – an RNA-seq expression database for polyploid wheat.

Webinars and presentations

• RNASeq-Blog Presentations
• RNA-Seq Workshop Documentation (UC Davis University)
• Princeton Workshop
• YouTube/RNA-Seq
• RNA-Seq Presentations from GSK, University of Torino and University of Bath.