RNA-seq

Tool	Year Published	Notable Features	Programming language	Package manager	Required expertise	Software	Type of URL 1. Web services designed to host source code 2. Others (e.g personal and/or university web services)
a. Data quality control
iSeqQC1	2020	Expression-based raw data QC tool that detects outliers	R	N/A	++	https://github.com/gkumar09/iSeqQC	1
qsmooth2	2018	Adaptive smooth quantile normalization	R	Bioconductor	++	http://bioconductor.org/packages/release/bioc/html/qsmooth.html	1
FastQC3	2018	Raw data QC tool for for high throughput sequence data	Java	Anaconda	++	https://github.com/s-andrews/fastqc/	1
QC34	2014	Raw data QC tool detecting batch effect and cross contamination	Perl, R	Anaconda	++	https://github.com/slzhao/QC3	1
kPAL5	2014	Alignment-free assessment raw data QC tool by analyzing k-mer frequencies	Python	Anaconda	++	https://github.com/LUMC/kPAL	1
HTQC6	2013	Raw data QC read assessment and filtration	C++	N/A	+++	https://sourceforge.net/projects/htqc/	1
Trimmomatic7	2014	Trimming of reads and removal of adapters	Java	Anaconda	++	http://www.usadellab.org/cms/index.php?page=trimmomatic	2
Skewer8	2014	Adapter trimming of reads	C++	Anaconda	++	https://sourceforge.net/projects/skewer	1
Flexbar9	2012	Trimming of reads and adaptor removal	C++	Anaconda	++	https://github.com/seqan/flexbar	1
QuaCRS10	2014	Post QC tool by performing meta-analyses on QC metrics across large numbers of samples.	Python	N/A	+++	https://github.com/kwkroll32/QuaCRS	1
BlackOPs11	2013	Post QC tool that simulates experimental RNA-seq derived from the reference genome and aligns these sequences and outputs a blacklist of positions and alleles caused by mismapping	Perl	N/A	+++	https://sourceforge.net/projects/rnaseqvariantbl/	1
RSeQC12	2012	Post QC evaluation of different aspects of RNA-seq experiments, such as sequence quality, GC bias, nucleotide composition bias, sequencing depth, strand specificity, coverage uniformity and read distribution over the genome structure.	Python, C	Anaconda	++	http://rseqc.sourceforge.net/	1
RNA-SeQC13	2012	RNA-seq metrics for post- quality control and process optimization	Java	Anaconda	++	https://software.broadinstitute.org/cancer/cga/rna-seqc	2
Seqbias14	2012	Post QC tool using a graphical model to increase accuracy of de novo gene annotation, uniformity of read coverage, consistency of nucleotide frequencies and agreement with qRT-PCR	R	Anaconda, Bioconductor	++	http://master.bioconductor.org/packages/devel/bioc/html/seqbias.html	1
SAMStat15	2011	Post QC tool which plotsPost nucleotide overrepresentation and other statistics in mapped and unmapped reads in a html page	C	N/A	+++	http://samstat.sourceforge.net	1
Samtools16	2009	Post QC tool using generic alignment format for storing read alignments against reference sequences and to visualize the Binary/Alignment Map (BAM).	C, Perl	Anaconda	+	https://github.com/samtools/samtools	1
b. Read alignment
deSALT17	2019	Long transcriptomic read alignment with de Bruijn graph-based index	C	Anaconda	++	https://github.com/ydLiu-HIT/deSALT	1
Magic-BLAST18	2018	Aligner for long and short reads through optimization of a spliced alignment score	C++	N/A	+++	https://ncbi.github.io/magicblast/	1
Minimap219	2018	Alignment using seed chain alignment procedure	C, Python	Anaconda	++	https://github.com/lh3/minimap2	1
DART20	2018	Burrows-Wheeler Transform based aligner which adopts partitioning strategy to divide a read into two groups	C/C++	Anaconda	++	https://github.com/hsinnan75/DART	1
MMR21	2016	Resolves the mapping location of multi-mapping reads, optimising for locally smooth coverage.	C++	N/A	+++	https://github.com/ratschlab/mmr	1
ContextMap 222	2015	Allows parallel mapping against several reference genomes	Java	N/A	+++	http://www.bio.ifi.lmu.de/ContextMap	2
HISAT23	2015	Aligning reads using an indexing scheme based on the Burrows-Wheeler transform and the Ferragina-Manzini (FM) index	C++	Anaconda	++	http://www.ccb.jhu.edu/software/hisat/index.shtml	2
Segemehl24	2014	Multi-split mapping for circular RNA, trans-splicing, and fusion events in addition to performing splice alignment	C, C++, Perl, Python, Shell (Bash)	Anaconda	++	(http://www.bioinf.uni-leipzig.de/Software/segemehl/).	2
JAGuaR25	2014	Uses a modified GTF (Gene Transfer Format) of known splice sites to build the complete sequence from all reads mapped to the transcript.	Python	N/A	+++	https://www.bcgsc.ca/resources/software/jaguar	2
CRAC26	2013	Uses double K-mer indexing and profiling approach to map reads, predict SNPs, gene fusions, repeat borders.	C++	Anaconda	++	http://crac.gforge.inria.fr/	2
STAR27	2013	Aligns long reads against genome reference database	C++	Anaconda	++	https://github.com/alexdobin/STAR	1
Subread28	2013	Mapping reads to a reference genome using multi-seed strategy, called seed-and-vote	C, R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/Rsubread.html	1
TopHat229	2013	Alignment of transcriptomes in the presence of insertions, deletions and gene fusions	C++, Python	Anaconda	++	http://ccb.jhu.edu/software/tophat/index.shtml	2
OSA30	2012	K-mer profiling approach to map reads	C#	N/A	+++	http://www.arrayserver.com/wiki/index.php?title=OSA	2
PASSion31	2012	Pattern growth pipeline for splice junction detection	C++, Perl, Shell (Bash)	N/A	+++	https://trac.nbic.nl/passion/	2
RUM32	2011	Comparative analysis of RNA-seq alignment algorithms and the RNA-seq unified mapper	Perl, Python	N/A	+++	http://www.cbil.upenn.edu/RUM/	2
SOAPSplice33	2011	Ab initio detection of splice junctions	Perl	Anaconda	++	http://soap.genomics.org.cn/soapsplice.html	2
MapSplice34	2010	De novo detection of splice junctions	C++	Anaconda	++	https://github.com/LiuBioinfo/MapSplice	1
SpliceMap35	2010	De novo detection of splice junctions and RNA-seq alignment	C++	Anaconda	++	http://web.stanford.edu/group/wonglab/SpliceMap/	2
Supersplat36	2010	De novo detection of splice junctions	C++	N/A	+++	http://mocklerlab.org/tools/1/manual	2
HMMSplicer37	2010	Detection of splice junctions of short sequence reads	Python	N/A	+++	http://derisilab.ucsf.edu/software/hmmsplicer	2
QPALMA38	2008	Spliced alignments of short sequence reads.	C++, Python	N/A	+++	http://www.raetschlab.org/suppl/qpalma	2
c. Gene annotations
SQANTI39	2018	Analyses quality of long reads transcriptomes and removes artefacts.	Python	Anaconda	++	https://github.com/ConesaLab/SQANTI	1
Annocript40	2015	Databases are downloaded to annotate protein coding transcripts with the prediction of putative long non-coding RNAs in whole transcriptomes.	Perl, Python, R	N/A	+++	https://github.com/frankMusacchia/Annocript	1
CIRI41	2015	De novo circular RNA identification	Perl	N/A	+++	https://sourceforge.net/projects/ciri/	1
TSSAR42	2014	Automated de novo TSS annotation from differential RNA-seq data	Java, Perl, R	Anaconda	++	http://rna.tbi.univie.ac.at/TSSAR	2
d. Transcriptome assembly
FLAIR43	2020	Full-length alternative isoform analysis of RNA	Python	Anaconda	++	https://github.com/BrooksLabUCSC/FLAIR	1
Scallop44	2017	Splice-graph-decomposition algorithm which optimizes two competing objectives while satisfying all phasing constraints posed by reads spanning multiple vertices	C++	Anaconda	+++	https://github.com/Kingsford-Group/scallop	1
CLASS245	2016	Splice variant annotation	C++, Perl, Shell	Anaconda	++	https://sourceforge.net/projects/splicebox/	1
StringTie46	2015	Applies a network flow algorithm originally developed in optimization theory, together with optional de novo assembly, to assemble transcripts	C++	N/A	+++	http://ccb.jhu.edu/software/stringtie	2
Bridger47	2015	De novo transcript assembler using a mathematical model, called the minimum path cover	C++, Perl	N/A	+++	https://sourceforge.net/projects/rnaseqassembly/files/?source=navbar	1
Bayesembler48	2014	Reference genome guided transcriptome assembly built on a Bayesian model	C++	N/A	+++	https://github.com/bioinformatics-centre/bayesembler.	1
SEECER49	2013	De novo transcriptome assembly using hidden Markov Model (HMM) based method	C++	N/A	+++	http://sb.cs.cmu.edu/seecer/	2
BRANCH50	2013	De novo transcriptome assemblies by using genomic information that can be partial or complete genome sequences from the same or a related organism.	C++	N/A	+++	https://github.com/baoe/BRANCH	1
EBARDenovo51	2013	De novo transcriptome assembly uses an efficient chimera-detection function	C#	N/A	+++	https://sourceforge.net/projects/ebardenovo/	1
Oases52	2012	De novo transcriptome assembly using k-mer profiling and building a de Brujin graph	C	N/A	+++	https://github.com/dzerbino/oases/tree/master	1
Cufflinks53	2012	Ab initio transcript assembly, estimates their abundances, and tests for differential expression	C++	N/A	+++	https://github.com/cole-trapnell-lab/cufflinks	1
IsoInfer54	2011	Infer isoforms from short reads	C/C++	N/A	+++	http://www.cs.ucr.edu/~jianxing/IsoInfer.html	2
IsoLasso55	2011	Reference genome guided using LASSO regression approach	C++	N/A	+++	http://alumni.cs.ucr.edu/~liw/isolasso.html	2
Trinity56	2011	De novo transcriptome assembly	C++, Java, Perl, R, Shell (Bash)	Anaconda	++	https://github.com/trinityrnaseq/trinityrnaseq/wiki	1
Trans-ABySS57	2010	De novo short-read transcriptome assembly and can also be used for fusion detection	Python	N/A	+++	https://github.com/bcgsc/transabyss	1
Scripture58	2010	Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells	Java	N/A	+++	www.broadinstitute.org/software/Scripture/	2
e. Transcriptome quantification
TALON59	2019	Long-read transcriptome discovery and quantification	Python	N/A	+++	https://github.com/dewyman/TALON	1
Salmon60	2017	Composed of: lightweight-mapping model, an online phase that estimates initial expression levels and model parameters, and an offline phase that refines expression estimates models, and mesures sequence-specific, fragment GC, and positional biases	C++	Anaconda	++	https://github.com/COMBINE-lab/Salmon	1
Kallisto61	2016	K-mer based pseudoalignment for allligment free transcript and gene expression quantification	C, C++, Perl	Anaconda	++	https://github.com/pachterlab/kallisto	1
Wub62	2016	Sequence and error simulation tool to calculate read and genome assembly accuracy.	Python	Anaconda	++	https://github.com/nanoporetech/wub	1
Rcount63	2015	GUI based tool used for quantification using counts per feature	Web based tool	N/A	+	https://github.com/MWSchmid/Rcount	1
Ht-seq64	2015	Calculates gene counts by counting number of reads overlapping genes	Python	pip	++	https://htseq.readthedocs.io/en/release_0.11.1/overview.html	2
EMSAR65	2015	Estimation by mappability-based segmentation and reclustering using a joint Poisson model	C	N/A	+++	https://github.com/parklab/emsar	1
Maxcounts66	2014	Quantify the expression assigned to an exon as the maximum of its per-base counts	C++	N/A	+++	http://sysbiobig.dei.unipd.it/?q=Software#MAXCOUNTS	2
FIXSEQ67	2014	A nonparametric and universal method for processing per-base sequencing read count data.	R	N/A	++	https://bitbucket.org/thashim/fixseq/src/master/	1
Sailfish68	2014	EM based quantification using statistical coupling between k-mers.	C, C++	Anaconda	++	https://github.com/kingsfordgroup/sailfish	1
Casper69	2014	Bayesian modeling framework to quantify alternative splicing.	R	Anaconda, Bioconductor	++	http://www.bioconductor.org/packages/release/bioc/html/casper.html	1
MaLTA70	2014	Simultaneous transcriptome assembly and quantification from Ion Torrent RNA-Seq data.	C++	N/A	+++	http://alan.cs.gsu.edu/NGS/?q=malta	2
Featurecounts71	2014	Read summarization program for counting reads generated.	R	N/A	++	http://subread.sourceforge.net/	1
MITIE72	2013	Transcript reconstruction and assembly from RNA-Seq data using mixed integer optimisation.	MATLAB, C++	N/A	+++	https://github.com/ratschlab/MiTie	1
iReckon73	2013	EM-based method to accurately estimate the abundances of known and novel isoforms.	Java	N/A	+++	http://compbio.cs.toronto.edu/ireckon/	2
eXpress74	2013	Online EM based algorithm for quantification which considers one read at a time.	C++, Shell (Bash)	Anaconda	++	https://pachterlab.github.io/eXpress/manual.html	1
BitSeq75	2012	Bayesian transcript expression quantification and differential expression.	C++, R	Anaconda,Bioconductor	++	http://bitseq.github.io/	1
IQSeq76	2012	Integrated isoform quantification analysis.	C++	N/A	+++	http://archive.gersteinlab.org/proj/rnaseq/IQSeq/	2
CEM77	2012	Statistical framework for both transcriptome assembly and isoform expression level estimation.	Python	N/A	+++	http://alumni.cs.ucr.edu/~liw/cem.html	2
SAMMate78	2011	Analysis of differential gene and isoform expression.	Java	N/A	+++	http://sammate.sourceforge.net/	1
Isoformex79	2011	Estimation method to estimate the expression levels of transcript isoforms.	N/A	N/A	N/A	http://bioinformatics.wistar.upenn.edu/isoformex	2
IsoEM80	2011	EM based method for inference of isoform and gene-specific expression levels	Java	N/A	+++	http://dna.engr.uconn.edu/software/IsoEM/.	2
RSEM81	2011	Ab initio EM based method for inference of isoform and gene-specific expression levels	C++, Perl, Python, R	Anaconda	++	https://github.com/deweylab/RSEM	1
EDASeq82	2011	Within-lane GC-content normalization, between-sample normalization, visualization.	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/devel/bioc/html/EDASeq.html	1
MMSEQ83	2011	Haplotype and isoform specific expression estimation	C++, R, Ruby, Shell (Bash)	N/A	++	https://github.com/eturro/mmseq	1
MISO84	2010	Statistical model that estimates expression of alternatively spliced exons and isoforms	C, Python	N/A	+++	https://miso.readthedocs.io/en/fastmiso/#latest-version-from-github	2
SOLAS85	2010	Prediction of alternative isoforms from exon expression levels	R	N/A	++	http://cmb.molgen.mpg.de/2ndGenerationSequencing/Solas/	2
Rseq86	2009	Statistical inferences for isoform expression	C++	N/A	+++	http://www-personal.umich.edu/~jianghui/rseq/#download	2
rQuant87	2009	Estimating density biases and considering the read coverages at each nucleotide independently using quadratic programming	Matlab, Shell (Bash), Javascript	N/A	+++	https://galaxy.inf.ethz.ch/?tool_id=rquantweb&version=2.2&__identifer=3iuqb8nb3wf	2
ERANGE88	2008	Mapping and quantifying mammalian transcripts	Python	N/A	+++	http://woldlab.caltech.edu/rnaseq	2
f. Differential expression
Swish89	2019	Non-parametric model for differential expression analysis using inferential replicate counts	R	Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/fishpond.html	1
Yanagi90	2019	Transcriptome segment analysis	Python/C++	N/A	+++	https://github.com/HCBravoLab/yanagi	1
Whippet91	2018	Quantification of transcriptome structure and gene expression analysis using EM.	Julia	N/A	+++	https://github.com/timbitz/Whippet.jl	1
ReQTL92	2018	Identifies correlations between SNVs and gene expression from RNA-seq data	R	N/A	++	https://github.com/HorvathLab/ReQTL	1
vast-tools93	2017	Profiling and comparing alternative splicing events in RNA-Seq data and for downstream analyses of alternative splicing.	R, Perl	N/A	+++	https://github.com/vastgroup/vast-tools	1
Ballgown94	2015	Linear model–based differential expression analyses	R	Anaconda, Bioconductor	++	https://github.com/alyssafrazee/ballgown	1
Limma/Voom95	2014	Linear model-based differential expression and differential splicing analyses	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/limma.html	1
rMATS96	2014	Detect major differential alternative splicing types in RNA-seq data with replicates.	Python, C++	Anaconda	++	http://rnaseq-mats.sourceforge.net/rmats3.2.5/	1
DESeq297	2014	Differential analysis of count data, using shrinkage estimation for dispersions and fold changes	R	Bioconductor, CRAN	++	https://bioconductor.org/packages/release/bioc/html/DESeq2.html	1
Corset98	2014	Differential gene expression analysis for de novo assembled transcriptomes	C++	Anaconda	++	https://github.com/Oshlack/Corset/wiki	1
BADGE99	2014	Bayesian model for accurate abundance quantification and differential analysis	Matlab	N/A	+++	http://www.cbil.ece.vt.edu/software.htm	2
compcodeR100	2014	Benchmarking of differential expression analysis methods	R	Anaconda, Bioconductor	++	https://www.bioconductor.org/packages/compcodeR/	1
metaRNASeq101	2014	Differential meta-analyses of RNA-seq data	R	Anaconda, CRAN	++	http://cran.r-project.org/web/packages/metaRNASeq	1
Characteristic Direction102	2014	Geometrical multivariate approach to identify differentially expressed genes	R, Python, MATLAB	N/A	++	http://www.maayanlab.net/CD	2
HTSFilter103	2013	Filter-replicated high-throughput transcriptome sequencing data	R	Anaconda, Bioconductor	++	http://www.bioconductor.org/packages/release/bioc/html/HTSFilter.html	1
NPEBSeq104	2013	Nonparametric empirical bayesian-based procedure for differential expression analysis	R	N/A	++	http://bioinformatics.wistar.upenn.edu/NPEBseq	2
EBSeq105	2013	Identifying differentially expressed isoforms.	R	Anaconda, Bioconductor	++	http://bioconductor.org/packages/release/bioc/html/EBSeq.html	1
sSeq106	2013	Shrinkage estimation of dispersion in Negative Binomial models	R	Anaconda, Bioconductor	++	http://bioconductor.org/packages/release/bioc/html/sSeq.html	1
Cuffdiff2107	2013	Differential analysis at transcript resolution	C++, Python	N/A	+++	http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/	1
SAMseq108	2013	Nonparametric method with resampling to account for the different sequencing depths	R	CRAN	++	https://rdrr.io/cran/samr/man/SAMseq.html	1
DSGseq 109	2013	NB-statistic method that can detect differentially spliced genes between two groups of samples without using a prior knowledge on the annotation of alternative splicing.	R	N/A	++	http://bioinfo.au.tsinghua.edu.cn/software/DSGseq/	2
NOISeq110	2011	Uses a non-parametric approach for differential expression analysis and can work in absence of replicates	R	Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/NOISeq.html	1
EdgeR111	2010	Examining differential expression of replicated count data and differential exon usage	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/edgeR.html	1
DEGseq112	2010	Identify differentially expressed genes or isoforms for RNA-seq data from different samples.	R	Bioconductor	++	http://bioconductor.org/packages/release/bioc/html/DEGseq.html	1
g. RNA splicing
LeafCutter113	2018	Detects differential splicing and maps quantitative trait loci (sQTLs).	R	Anaconda	++	https://github.com/davidaknowles/leafcutter.git	1
MAJIQ-SPEL114	2018	Visualization, interpretation, and experimental validation of both classical and complex splicing variation and automated RT-PCR primer design.	C++, Python	N/A	+++	https://galaxy.biociphers.org/galaxy/root?tool_id=majiq_spel	2
MAJIQ115	2016	Web-tool that takes as input local splicing variations (LSVs) quantified from RNA-seq data and provides users with a visualization package (VOILA) and quantification of gene isoforms.	C++, Python	N/A	+++	https://majiq.biociphers.org/commercial.php	2
SplAdder116	2016	Identification, quantification, and testing of alternative splicing events	Python	PyPI	+++	http://github.com/ratschlab/spladder	1
SplicePie117	2015	Detection of alternative, non-sequential and recursive splicing	Perl, R	N/A	+++	https://github.com/pulyakhina/splicing_analysis_pipeline	1
SUPPA118	2015	Alternative splicing analysis	Python, R	Anaconda	++	https://github.com/comprna/SUPPA	1
SNPlice119	2015	Identifying variants that modulate Intron retention	Python	N/A	+++	https://code.google.com/p/snplice/	1
IUTA120	2014	Detecting differential isoform usage	R	N/A	++	http://www.niehs.nih.gov/research/resources/software/biostatistics/iuta/index.cfm.	1
SigFuge121	2014	Identifying genomic loci exhibiting differential transcription patterns	R	Anaconda, Bioconductor	++	http://bioconductor.org/packages/release/bioc/html/SigFuge.html	1
FineSplice122	2014	Splice junction detection and quantification	Python	N/A	+++	https://sourceforge.net/p/finesplice/	1
PennSeq123	2014	Statistical method that allows each isoform to have its own non-uniform read distribution	Perl	N/A	+++	http://sourceforge.net/projects/pennseq	1
FlipFlop124	2014	RNA isoform identification and quantification with network flows	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/flipflop.html	1
GESS125	2014	Graph-based exon-skipping scanner for de novo detection of skipping event sites	N/A	N/A	N/A	http://jinlab.net/GESS_Web/	2
spliceR126	2013	Classification of alternative splicing and prediction of coding potential	R	Anaconda, Bioconductor	++	http://www.bioconductor.org/packages/2.13/bioc/html/spliceR.html	1
RNASeq-MATS127	2013	Detects and analyzes differential alternative splicing events	C, Python	N/A	+++	http://rnaseq-mats.sourceforge.net/	1
SplicingCompass128	2013	Differential splicing detection	R	N/A	++	http://www.ichip.de/softwa	2
DiffSplice129	2013	Genome-wide detection of differential splicing	C++	N/A	+++	http://www.netlab.uky.edu/p/bioinfo/DiffSplice	2
DEXSeq130	2012	Statistical method to test for differential exon usage.	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/DEXSeq.html	1
SpliceSeq131	2012	Identifies differential splicing events between test and control groups.	Java	N/A	+++	http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview.	2
JuncBASE132	2011	Identification and quantification of alternative splicing, including unannotated splicing	Python	N/A	+++	https://github.com/anbrooks/juncBASE	1
ALEXA-seq133	2010	Alternative expression analysis.	Perl, R, Shell (Bash)	N/A	+++	http://www.alexaplatform.org/alexa_seq/.	1
h. Cell deconvolution
TIMER2.0134	2020	Web server for comprehensive analysis of Tumor-Infiltrating Immune Cells.	Web-tool R, Javascript	N/A	+	https://github.com/taiwenli/TIMER	1
CIBERSORTx135	2019	Impute gene expression profiles and provide an estimation of the abundances of member cell types in a mixed cell population.	Web-tool Java, R	N/A	+	https://cibersortx.stanford.edu/	2
quanTIseq136	2019	Quantify the fractions of ten immune cell types from bulk RNA-sequencing data.	R, Shell (Bash)	DockerHub	+	https://icbi.i-med.ac.at/quantiseq	2
Immunedeconv137	2019	Benchmarking of transcriptome-based cell-type quantification methods for immuno-oncology	R	Anaconda	++	https://github.com/icbi-lab/immunedeconv	1
Linseed138	2019	Deconvolution of cellular mixtures based on linearity of transcriptional signatures.	C++, R	N/A	++	https://github.com/ctlab/LinSeed	1
deconvSEQ139	2019	Deconvolution of cell mixture distribution based on a generalized linear model.	R	N/A	++	https://github.com/rosedu1/deconvSeq	1
CDSeq140	2019	Simultaneously estimate both cell-type proportions and cell-type-specific expression profiles.	MATLAB, R	N/A	++	https://github.com/kkang7/CDSeq_R_Package	1
Dtangle141	2019	Estimates cell type proportions using publicly available, often cross-platform, reference data.	R	Anaconda,CRAN	++	https://github.com/gjhunt/dtangle	1
GEDIT142	2019	Estimate cell type abundances.	Web based tool Python, R	N/A	+	http://webtools.mcdb.ucla.edu/	2
SaVant143	2017	Web based tool for sample level visualization of molecular signatures in gene expression profiles.	Javascript, R	N/A	+++	http://newpathways.mcdb.ucla.edu/savant	2
EPIC144	2017	Simultaneously estimates the fraction of cancer and immune cell types.	R	N/A	++	http://epic.gfellerlab.org/	2
WSCUnmix145	2017	Automated deconvolution of structured mixtures.	MATLAB	N/A	+++	https://github.com/tedroman/WSCUnmix	1
Infino146	2017	Deconvolves bulk RNA-seq into cell type abundances and captures gene expression variability in a Bayesian model to measure deconvolution uncertainty.	R, Python	Docker Hub	++	https://github.com/hammerlab/infino	1
MCP-counter147	2016	Estimating the population abundance of tissue-infiltrating immune and stromal cell populations.	R	N/A	+	https://github.com/ebecht/MCPcounter	1
CellCode148	2015	Latent variable approach to differential expression analysis for heterogeneous cell populations.	R	N/A	++	http://www.pitt.edu/~mchikina/CellCODE/	2
PERT149	2012	Probabilistic expression deconvolution method.	MATLAB	N/A	+++	https://github.com/gquon/PERT	1
i. Immune repertoire profiling
ImReP150	2018	Profiling immunoglobulin repertoires across multiple human tissues.	Python	N/A	+++	https://github.com/mandricigor/imrep/wiki	1
TRUST (T cell)151	2016	Landscape of tumor-infiltrating T cell repertoire of human cancers.	Perl	N/A	+++	https://github.com/liulab-dfci/TRUST4	1
V’DJer 152	2016	Assembly-based inference of B-cell receptor repertoires from short reads with V’DJer.	C, C++	Anaconda	++	https://github.com/mozack/vdjer	1
IgBlast-based pipeline153	2016	Statistical inference of a convergent antibody repertoire response.	C++	N/A	+++	https://www.ncbi.nlm.nih.gov/igblast/	1
MiXCR154	2015	Processes big immunome data from raw sequences to quantitated clonotypes.	Java	Anaconda	++	https://github.com/milaboratory/mixcr	1
j. Allele specific expression
EAGLE155	2017	Bayesian model for identifying GxE interactions based on associations between environmental variables and allele-specific expression.	C++, R	N/A	++	https://github.com/davidaknowles/eagle	1
ANEVA-DOT/ANEVA156	2019	Identify ASE outlier genes / Quantify genetic variation in gene dosage from ASE data.	R	N/A	++	https://github.com/PejLab/ANEVA-DOT	1
aFC157	2017	Quantifying the regulatory effect size of cis-acting genetic variation	Python	N/A	+++	https://github.com/secastel/aFC	1
phASER158	2016	Uses readback phasing to produce haplotype level ASE data (as opposed to SNP level)	Python	N/A	+++	https://github.com/secastel/phaser	1
RASQUAL159	2016	Maps QTLs for sequenced based cellular traits by combining population and allele-specific signals.	C, R	N/A	+++	https://github.com/natsuhiko/rasqual	1
allelecounter160	2015	Generate ASE data from RNAseq data and a genotype file.	Python	N/A	+++	https://github.com/secastel/allelecounter	1
WASP161	2015	Unbiased allele-specific read mapping and discovery of molecular QTLs	C Python	Anaconda	++	https://github.com/bmvdgeijn/WASP/	1
Mamba162	2015	Compares different patterns of ASE across tissues	R	N/A	++	http://www.well.ox.ac.uk/~rivas/mamba/.	2
MBASED 163	2014	Allele-specific expression detection in cancer tissues and cell lines	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/MBASED.html	1
Allim164	2013	Estimates allele-specific gene expression.	Python, R	N/A	+++	https://sourceforge.net/projects/allim/	1
AlleleSeq165	2011	Identifies allele-specific events in mapped reads between maternal and paternal alleles.	Python, Shell	N/A	+++	http://alleleseq.gersteinlab.org/	2
k. Viral detection
ROP166	2018	Dumpster diving in RNA-sequencing to find the source of 1 trillion reads across diverse adult human tissues	Python, Shell (Bash)	Anaconda	++	https://github.com/smangul1/rop	1
RNA CoMPASS167	2014	Simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens.	Perl, Shell, Java	N/A	++	http://rnacompass.sourceforge.net/	1
VirusSeq168	2013	Identify viruses and their integration sites using next-generation sequencing of human cancer tissues	Perl, Shell (Bash)	N/A	+++	http://odin.mdacc.tmc.edu/∼xsu1/VirusSeq.html	2
VirusFinder169	2013	Detection of Viruses and Their Integration Sites in Host Genomes through Next Generation Sequencing Data	Perl	N/A	+++	http://bioinfo.mc.vanderbilt.edu/VirusFinder/	2
l. Fusion detection
INTEGRATE-Vis170	2017	Generates plots focused on annotating each gene fusion at the transcript- and protein-level and assessing expression across samples.	Python	N/A	+++	https://github.com/ChrisMaherLab/INTEGRATE-Vis	1
INTEGRATE-Neo171	2017	Gene fusion neoantigen discovery tool, which uses RNA-Seq reads and is capable of reporting tumor-specific peptides recognizable by immune molecules.	Python, C++	N/A	+++	https://github.com/ChrisMaherLab/INTEGRATE-Neo	1
INTEGRATE172	2016	Capable of integrating aligned RNA-seq and WGS reads and characterizes the quality of predictions.	C++	N/A	+++	https://sourceforge.net/projects/integrate-fusion/	1
TRUP173	2015	Combines split-read and read-pair analysis with de novo regional assembly for the identification of chimeric transcripts in cancer specimens.	C++, Perl, R	N/A	+++	https://github.com/ruping/TRUP	1
PRADA174	2014	Detect gene fusions but also performs alignments, transcriptome quantification; mainly integrated genome/transcriptome read mapping.	Python	Anaconda	++	http://sourceforge.net/projects/prada/	1
Pegasus175	2014	Annotation and prediction of biologically functional gene fusion candidates.	Java, Perl, Python, Shell (Bash)	N/A	+++	https://github.com/RabadanLab/Pegasus	1
FusionCatcher176	2014	Finding somatic fusion genes	Python	Anaconda	++	https://sourceforge.net/projects/fusioncatcher/	1
FusionQ177	2013	Gene fusion detection and quantification from paired-end RNA-seq	C++, Perl, R	N/A	+++	http://www.wakehealth.edu/CTSB/Software/Software.htm	2
Barnacle178	2013	Detecting and characterizing tandem duplications and fusions in de novo transcriptome assemblies	Python, Perl	N/A	+++	http://www.bcgsc.ca/platform/bioinfo/software/barnacle	2
Dissect179	2012	Detection and characterization of structural alterations in transcribed sequences	C	N/A	+++	http://dissect-trans.sourceforge.net	1
BreakFusion180	2012	Targeted assembly-based identification of gene fusions	C++, Perl	N/A	+++	https://bioinformatics.mdanderson.org/public-software/breakfusion/	2
EricScript181	2012	Identification of gene fusion products in paired-end RNA-seq data.	Perl, R, Shell (Bash)	Anaconda	++	http://ericscript.sourceforge.net	1
Bellerophontes182	2012	Chimeric transcripts discovery based on fusion model.	Java, Perl, Shell (Bash)	N/A	+++	http://eda.polito.it/bellerophontes/	2
GFML183	2012	Standard format for organizing and representing the significant features of gene fusion data.	XML	N/A	+	http://code.google.com/p/gfml-prototype/	1
FusionHunter184	2011	Identifies fusion transcripts from transcriptional analysis.	C++	N/A	+++	https://github.com/ma-compbio/FusionHunter	1
ChimeraScan185	2011	Identifying chimeric transcription.	Python	Anaconda	++	https://code.google.com/archive/p/chimerascan/downloads	1
TopHat-fusion186	2011	Discovery of novel fusion transcripts.	C++, Python	N/A	+++	http://ccb.jhu.edu/software/tophat/fusion_index.shtml	2
deFuse187	2011	Fusion discovery in tumor RNA-seq data.	C++, Perl, R	Anaconda	++	https://github.com/amcpherson/defuse/blob/master/README.md	1
m. Detecting circRNA
CIRIquant188	2020	Accurate quantification and differential expression analysis of circRNAs.	Python	PyPI	++	https://sourceforge.net/projects/ciri/files/CIRIquant	1
CIRI-vis189	2020	Visualization of circRNA structures.	Java	N/A	+++	https://sourceforge.net/projects/ciri/files/CIRI-vis	1
Ularcirc190	2019	Analysis and visualisation of canonical and back splice junctions.	R	Bioconductor	++	https://github.com/VCCRI/Ularcirc	1
CLEAR191	2019	Circular and Linear RNA expression analysis.	Python	N/A	+++	https://github.com/YangLab/CLEAR	1
CIRI-full192	2019	Reconstruct and quantify full-length circular RNAs.	Java	N/A	+++	https://sourceforge.net/projects/ciri/files/CIRI-full	1
circAST193	2019	Full-length assembly and quantification of alternatively spliced isoforms in Circular RNAs	Python	N/A	+++	https://github.com/xiaofengsong/CircAST	1
CIRI2194	2018	Denovo circRNA identification	Pearl	N/A	+++	https://sourceforge.net/projects/ciri/files/CIRI2	1
Sailfish-cir195	2017	Quantification of circRNAs using model-based framework	Python	N/A	+++	https://github.com/zerodel/sailfish-cir	1
CircComPara196	2017	Multi‐method detection of circRNAs	R, Python	N/A	+++	http://github.com/egaffo/CirComPara	1
UROBORUS197	2016	Computationally identifying circRNAs from RNA-seq data	Perl	N/A	+++	https://github.com/WGLab/UROBORUS/tree/master/bin	1
PTESFinder198	2016	Identification of non-co-linear transcripts	Shell, Java	N/A	+++	https://sourceforge.net/projects/ptesfinder-v1/	1
NCLscan199	2016	identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA)	Python	N/A	+++	https://github.com/TreesLab/NCLscan	1
DCC200	2016	Specific identification and quantification of circRNA	Python	N/A	+++	https://github.com/dieterich-lab/DCC	1
CIRI-AS201	2016	Identification of internal structure and alternative splicing events in circRNA	Perl	N/A	+++	https://sourceforge.net/projects/ciri/files/CIRI-AS	1
circTest200	2016	Differential expression analysis and plotting of circRNAs	R	N/A	+++	https://github.com/dieterich-lab/CircTest	1
CIRCexplorer2202	2016	Annotation and de novo assembly of circRNAs	Python	Anaconda	++	https://github.com/YangLab/CIRCexplorer2	1
KNIFE 203	2015	Statistically based detection of circular and linear isoforms from RNA-seq data	Perl, Python, R	N/A	+++	https://github.com/lindaszabo/KNIFE	1
circRNA_finder204	2014	Identification of circRNAs from RNA-seq data	Perl	N/A	+++	https://github.com/orzechoj/circRNA_finder	1
find_circ205	2013	Identification of circRNAs based on head-to-tail spliced sequencing reads	Python	PyPI	++	https://github.com/marvin-jens/find_circ	1
n. Small RNA detection
miRTrace206	2018	Quality control of miRNA-seq data, identifies cross-species contamination.	Java	Anaconda	+++	https://github.com/friedlanderlab/mirtrace	1
sRNAbench207	2015	Expression profiling of small RNAs, prediction of novel microRNAs, analysis of isomiRs, genome mapping and read length statistics.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/srnabench/	2
sRNAde207	2015	Detection of differentially expressed small RNAs based on three programs.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/srnade/	2
sRNAblast207	2015	Aimed to determine the origin of unmapped or unassigned reads by means of a blast search against several remote databases.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/srnablast/	2
miRNAconsTarget207	2015	Consensus target prediction on user provided input data.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/amirconstarget/	2
sRNAjBrowser207	2015	Visualization of sRNA expression data in a genome context.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/srnajbrowser/	2
sRNAjBrowserDE207	2015	Visualization of differential expression as a function of read length in a genome context.	Web based tool	N/A	+	https://bioinfo5.ugr.es/srnatoolbox/srnajbrowserde/	2
ShortStack208	2013	Analyzes reference-aligned sRNA-seq data and performs. comprehensive de novo annotation and quantification of the inferred sRNA genes.	Perl	Anaconda	+++	https://github.com/MikeAxtell/ShortStack	1
mirTools 2.0209	2013	Detect, identify and profile various types, functional annotation and differentially expressed sRNAs.	Web based tool	N/A	+	http://www.wzgenomics.cn/mr2_dev/	2
UEA sRNA Workbench210	2012	Complete analysis of single or multiple-sample small RNA datasets.	Web based tool, C++, Java	N/A	+++	https://sourceforge.net/projects/srnaworkbench/	1
miRDeep2211	2011	Discovers known and novel miRNAs, quantifies miRNA expression.	Perl	Anaconda	+++	https://github.com/rajewsky-lab/mirdeep2	1
miRanalyzer212	2011	Detection of known and prediction of new microRNAs in high-throughput sequencing experiments.	Web based tool	N/A	+	http://bioinfo2.ugr.es/miRanalyzer/miRanalyzer.php	2
SeqBuster213	2010	Provides an automatized pre-analysis for sequence annotation for analysing small RNA data from Illumina sequencing.	Web based tool	Anaconda	+	http://estivill_lab.crg.es/seqbuster	2
DARIO214	2010	Allows to study short read data and provides a wide range of analysis features, including quality control, read normalization, and quantification.	Web based tool	N/A	+	http://dario.bioinf.uni-leipzig.de/index.py	2
o. Visualization tools
BEAVR215	2020	Facilitates interactive analysis and exploration of RNA-seq data, allowing statistical testing and visualization of the table of differentially expressed genes obtained.	R	Docker Hub	++	https://github.com/developerpiru/BEAVR	1
coseq216	2018	Co-expression analysis of sequencing data	R	Anaconda, Bioconductor	++	https://bioconductor.org/packages/release/bioc/html/coseq.html	1
ReadXplorer217	2016	Read mapping analysis and visualization	Java	N/A	+++	https://www.uni-giessen.de/fbz/fb08/Inst/bioinformatik/software/ReadXplorer	2
Integrated Genome Browser218	2016	An interactive tool for visually analyzing tiling array data and enables quantification of alternative splicing	Java	N/A	+++	http://www.bioviz.org/	2
Sashimi plots219	2015	Quantitative visualization comparison of exon usage	Python	N/A	++	http://miso.readthedocs.org/en/fastmiso/sashimi.html	2
ASTALAVISTA220	2015	Reports all alternative splicing events reflected by transcript annotations	Java	Anaconda	++	http://astalavista.sammeth.net/	2
RNASeqBrowser221	2015	Incorporates and extends the functionality of the UCSC genome browser	Java	N/A	+++	http://www.australianprostatecentre.org/research/software/rnaseqbrowser	2
SplicePlot222	2014	Visualizing splicing quantitative trait loci	Python	N/A	+++	http://montgomerylab.stanford.edu/spliceplot/index.html	2
RNASeqViewer223	2014	Compare gene expression and alternative splicing	Python	N/A	+++	https://sourceforge.net/projects/rnaseqbrowser/	1
PrimerSeq224	2014	Systematic design and visualization of RT-PCR primers using RNA seq data	Java, C++, Python	N/A	+++	http://primerseq.sourceforge.net/	1
Epiviz225	2014	Combining algorithmic-statistical analysis and interactive visualization	R	Anaconda, Bioconductor	++	https://epiviz.github.io/	1
RNAbrowse226	2014	RNA-seq De Novo Assembly Results Browser	N/A	N/A	N/A	http://bioinfo.genotoul.fr/RNAbrowse	2
ZENBU227	2014	Interactive visualization and analysis of large-scale sequencing datasets	C++, Javascript	N/A	+	https://fantom.gsc.riken.jp/zenbu/	2
CummeRbund53	2012	Navigate through data produced from a Cuffdiff RNA-seq differential expression analysis	R	Anaconda, Bioconductor	++	http://bioconductor.org/packages/devel/bioc/html/cummeRbund.html	1
Splicing Viewer228	2012	Visualization of splice junctions and alternative splicing	Java	N/A	+++	http://bioinformatics.zj.cn/splicingviewer.	2

Table 1: Landscape of current computational methods for RNA-seq analysis. We categorized RNA-seq tools published from 2008 to 2020 based on processes in the RNA-seq pipeline and workflow; starting with data quality control, read alignment, gene annotations, transcriptome assembly, transcriptome quantification, differential expression, RNA splicing, cell deconvolution, immune repertoire profiling, allele specific expression, viral detection, fusion detection, detecting circRNA, small RNA detection, and visualization tools. The third column ("Notable Features") presents key functionalities and methods used. The fourth column ("Programing Language") presents the interface mode (e.g., GUI, web-based, programming language). The fifth column ("Package Manager") highlights if a package manager such as Anaconda, Bioconductor, CRAN, Docker Hub, pip, or PyPI is available for the tool. We designated the assumed expertise level with a +, ++, or +++ in the sixth column ("Required Expertise"). A "+" represents little to no required expertise which would be assigned to a GUI based/web interface tool. "++" was assigned to tools that require R and/or multiple programming languages and whose software is located on Anaconda, Bioconductor, CRAN, Docker Hub, pip, or PyPI. "+++" was assigned to tools that require expertise in languages such as C, C++, Java, Python, Perl, or Shell (Bash) and may or may not have a package manager present. For each tool, we provide the links where the published tool software can be found and downloaded ("Software"). In the seventh column ("Type of URL"), each tool was assigned a "1" for web services designed to host source code or "2" for others (e.g personal and/or university web services).

Methods

Determine features of RNA-seq tools

We compiled 228 RNA-seq tools published between 2008 and 2020 which have varying purposes and capabilities based on the type of analysis one is conducting or the biological questions one is answering. We have considered 15 domains of RNA-seq analysis (data quality control, read alignment, gene annotations, transcriptome assembly, transcriptome quantification, differential expression, RNA splicing, cell deconvolution, immune repertoire profiling, allele specific expression, viral detection, fusion detection, detecting circRNA, small RNA detection, and visualization tools). After assigning each tool a category based on its area of application, we highlighted each tool's ("Notable Features"). These notable features encompassed a range of functionalities: purpose of the tool, which features made the tool unique or not unique within its category and the way in which the tool would take RNA-seq data as an input and present an output. We documented whether each tool was web-based or required one or many programming languages for installation and/or utilization ("Programming Language"). In addition to the programming languages, we highlighted whether a package manager (e.g., Anaconda, Bioconductor, CRAN, Docker Hub, pip, or PyPI) was available. Based on the combination of which programming language was required and which package manager was available for each tool, we assessed the required expertise needed to be able to install or run the tool. If the tool was a web-based tool with no package manager available or a web-based tool along with programming languages and package manager present, we assigned the tool the little to none required expertise of "+". If the tool required only R as a programming language or along with other programming languages and had a package manager present, the tool was assigned a required expertise of "++". In addition, if programming languages other than R were required, and a package manager was present, the tool was also assigned a "++". Lastly, for tools that required a single programming language (other than R) or multiple programming languages required and presence of no package manager were assigned a "+++" for the most required expertise. Each published tool had a designated software link where the tool can be downloaded and installed. Based on the type of URL of the software links, we assigned the tools a "1" or "2". An assignment of "1" meant that the tool's software was hosted on a web service designed to host source code. An assignment of "2" meant that the tool's software was hosted on other web services (e.g personal and/or university web services).

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