# Appendix V. Software and Tools
## 0) Process files in different format
### 0.1) sequence
* [seqtk](https://github.com/lh3/seqtk)
* [bbmap](https://sourceforge.net/projects/bbmap/)
### 0.2) alignment
* [gffread](https://github.com/gpertea/gffread)
* [samtools](http://www.htslib.org/)
* [bamtools](https://github.com/pezmaster31/bamtools)
### 0.3) interval
* [bedtools](https://bedtools.readthedocs.io/en/latest/)
* [bedtk](https://github.com/lh3/bedtk)
## 1) Homolog analysis
### 1.1) Sequence based search
* [blast](https://blast.ncbi.nlm.nih.gov/Blast.cgi): 方便的网页工具
* [blat](https://genome.ucsc.edu/cgi-bin/hgBlat): a blast like tool
* [mmseqs](https://github.com/soedinglab/MMseqs2): 比blast更现代的同源搜索工具，推荐本地进行大量计算时使用
* [diamond](https://github.com/bbuchfink/diamond): 蛋白的同源搜索工具
### 1.2) Profile based search
* [hmmer](http://hmmer.org/): profile hmm based search for protein and nucleotide sequence
* [infernal](http://eddylab.org/infernal/): profile SCFG based search for structured noncoding RNA
* [hh-suite](https://github.com/soedinglab/hh-suite): profile hmm to profile hmm alignment
### 1.3) Multiple sequence alignment
* [MAFFT](https://mafft.cbrc.jp/alignment/software/)
* [clustal](http://www.clustal.org/)
* [T-Coffee](https://tcoffee.crg.eu/)
## 2) Genome Browsers
* [UCSD Genome Browser](https://genome.ucsc.edu/) ([@youtube](https://youtu.be/eTgEtfI65hA) [@bilibili](https://player.bilibili.com/player.html?aid=30448417\&cid=53132461\&page=1))
* [IGV](http://software.broadinstitute.org/software/igv/) ([@youtube](https://youtu.be/6_1ZcVw7ptU) [@bilibili](https://player.bilibili.com/player.html?aid=30448472\&cid=53133093\&page=1))
> see more in [our Tutorial](/teaching/part-iii.-ngs-data-analyses/1.mapping/1.1-genome-browser.md)
## 3) DNA-seq
### (3.1) Mapping and QC
* **Remove adaptor**
* [cutadapt](https://cutadapt.readthedocs.io/en/stable/)
* [TrimGalore](https://github.com/FelixKrueger/TrimGalore): 对cutadapt进行封装，自动识别常见adaptor
* [Trimmomatic](http://www.usadellab.org/cms/?page=trimmomatic)
* [fastp](https://github.com/OpenGene/fastp)
* **Mapping**
* [bowtie2](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
* [bowtie](https://bowtie-bio.sourceforge.net/manual.shtml)
* [bwa](https://bio-bwa.sourceforge.net/)
* **QC**
* [fastqc](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
### (3.2) Variant Calling
* **Mutation discovery**
* [GATK](https://gatk.broadinstitute.org/hc/en-us)
* [Varscan](http://dkoboldt.github.io/varscan/)
* **Mutation annotation**
* [ANNOVAR](http://annovar.openbioinformatics.org/en/latest/user-guide/download/)
### (3.3) Assembly
**denovo assembly software**
* [SPAdes](https://github.com/ablab/spades)
* the sub-utility metaSPAdes is designed for metagenome assembly
* [megahit](https://github.com/voutcn/megahit): designed for metagenome assembly
### (3.4) CNV
* **Whole genome Seq**
* [Control-FREEC](http://boevalab.inf.ethz.ch/FREEC/)
* **Whole exome Seq**
* [CONTRA](http://contra-cnv.sourceforge.net/)
* [ExomeCNV](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179661/)
### (3.5) SV (structural variation)
* **structural variation**
* [lumpy](https://github.com/arq5x/lumpy-sv)
* [Breakdancer](http://breakdancer.sourceforge.net/)
## 4) RNA-seq
### (4.1) RNA-seq
* **Mapping**
* [STAR](https://github.com/alexdobin/STAR)
* The sub-utility STARsolo is designed for mapping of single cell RNA-seq data
* [hisat2](http://daehwankimlab.github.io/hisat2/)
* **Expression Quantification**
* [featureCounts](http://subread.sourceforge.net/)
* [htseq-count](https://htseq.readthedocs.io/en/master/)
* [salmon](https://combine-lab.github.io/salmon/)
* [kallisto](https://pachterlab.github.io/kallisto/)
* **Differential Analysis**
* [deseq2](https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
* [edgeR](https://bioconductor.org/packages/release/bioc/html/edgeR.html)
* [limma](https://bioconductor.org/packages/release/bioc/html/limma.html)
* **Alternative Splicing Analysis**:
* [rMATS](http://rnaseq-mats.sourceforge.net/)
* [MAJIQ](https://majiq.biociphers.org/)
* [SUPPA](https://github.com/comprna/SUPPA)
* [DEXSeq](https://bioconductor.org/packages/release/bioc/html/DEXSeq.html)
* **RNA Editing**
* [RNAEditor](http://rnaeditor.uni-frankfurt.de/)
* [REDItools](http://code.google.com/p/reditools/)
### (4.2) Single Cell RNA-seq (scRNA-seq)
* [awesome-single-cell](https://github.com/seandavi/awesome-single-cell): a collection of single cell analysis tools
* [seurat](https://satijalab.org/seurat/): a widely used R package
* [scanpy](https://scanpy.readthedocs.io/en/stable/): a widely used python package
* [monocle](http://cole-trapnell-lab.github.io/monocle-release/): Trajectory analysis
* [cellphonedb](https://www.cellphonedb.org/): Cell-cell interaction analysis
* [scenic](https://scenic.aertslab.org/): Transcriptional regulatory network
* Tutorials
*
*
> [Nature Biotechnology 2020 38(3):254-257](https://www.nature.com/articles/s41587-020-0449-8)
| Software name | Developer | Price structure | Platform-specific | Relevant stages of experiment |
| -------------------------------------------------------------------------------------------------------------------------------------------------------- | ---------------------------- | --------------- | --------------------------- | ------------------------------------------------------------------------------------------------------------------------------------ |
| [Cell Ranger](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) | 10X Genomics | Free download | 10X Chromium | Raw read alignment, QC and matrix generation for scRNA-seq and ATAC-seq; data normalization; dimensionality reduction and clustering |
| [Loupe Cell Browser](https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser) | 10X Genomics | Free download | 10X Chromium | Visualization and analysis |
| [Partek Flow](https://www.partek.com/application-page/single-cell-gene-expression/) | Partek | License | No | Complete data analysis and visualization pipeline for scRNA-seq data |
| [Qlucore Omics Explorer](https://www.qlucore.com/single-cell-rnaseq) | Qlucore | License | No | scRNA-seq data filtering, dimensionality reduction and clustering, visualization |
| [mappa Analysis Pipeline](https://www.takarabio.com/products/automation-systems/icell8-system-and-software/bioinformatics-tools/mappa-analysis-pipeline) | Takara Bio | Free download | Takara ICell8 | Raw read alignment and matrix generation for scRNA-seq |
| [hanta R kit](https://www.takarabio.com/products/automation-systems/icell8-system-and-software/bioinformatics-tools/hanta-r-kit) | Takara Bio | Free download | Takara ICell8 | Clustering and analysis of mappa data |
| [Singular Analysis Toolset](https://www.fluidigm.com/software) | Fluidigm | Free download | Fluidigm C1 or Biomark | Analysis and visualization of differential gene expression data for scRNA-seq |
| [SeqGeq](https://www.flowjo.com/solutions/seqgeq) | FlowJo/BD Biosciences | License | No | Data normalization and QC, dimensionality reduction and clustering, analysis and visualization |
| [Seven Bridges](https://www.sevenbridges.com/bdgenomics/) | Seven Bridges/BD Biosciences | License | BD Rhapsody and Precise | Cloud-based raw read alignment, QC and matrix generation |
| [Tapestri Pipeline/Insights](https://missionbio.com/panels/software/) | Mission Bio | Free download | Mission Bio Tapestri | Analysis of single-cell genomics data |
| [BaseSpace SureCell](https://www.illumina.com/products/by-type/informatics-products/basespace-sequence-hub.html) | Illumina | License | Illumina SureCell libraries | Raw read alignment and matrix generation |
| [OmicSoft Array Studio](https://omicsoftdocs.github.io/ArraySuiteDoc/tutorials/scRNAseq/Introduction/) | Qiagen | License | No | Raw read alignment, QC and matrix generation, dimensionality reduction and clustering |
### 4.3 Assembly
* [Trinity](https://github.com/trinityrnaseq/trinityrnaseq/wiki): 利用RNA-seq数据进行转录本组装
## 5) Interactome
### **(5.1) ChIP-seq**
* [MACS](https://github.com/macs3-project/MACS): peak calling
* [homer](http://homer.ucsd.edu/homer/): peak calling, motif finding, etc
* [ChIPseeker](https://bioconductor.org/packages/release/bioc/html/ChIPseeker.html): visualization and annotation
### **(5.2) CLIP-seq**
* [CTK](https://zhanglab.c2b2.columbia.edu/index.php/CTK_Documentation)
* [Piranha](http://smithlabresearch.org/software/piranha/)
* [PARalyzer](https://ohlerlab.mdc-berlin.de/software/PARalyzer_85/)
* [clipper](https://github.com/YeoLab/clipper)
### **(5.3) Motif analysis**
**sequence**
1. MEME motif based sequence analysis tools
2. HOMER Software for motif discovery and next-gen sequencing analysis
**structure**
1. RNApromo Computational prediction of RNA structural motifs involved in post transcriptional regulatory processes
2. GraphProt modeling binding preferences of RNA-binding proteins
## 6) Epigenetic Data
### **(6.1) ChIP-seq**
* **Bisulfate sequencing**:
* Review: [Katarzyna Wreczycka, et al. Strategies for analyzing bisulfite sequencing data. Journal of Biotechnology. 2017.](https://www.sciencedirect.com/science/article/pii/S0168165617315936)
* Mapping:
* [Bismark](http://www.bioinformatics.babraham.ac.uk/projects/bismark/)
* [BSMAP](https://github.com/zyndagj/BSMAPz)
* Differential Methylation Regions (DMRs) detection
* [methylkit](https://bioconductor.org/packages/release/bioc/html/methylKit.html)
* [ComMet](https://github.com/yutaka-saito/ComMet)
* Segmentation of the methylome, Classification of Fully Methylated Regions (FMRs), Unmethylated Regions (UMRs) and Low-Methylated Regions (LMRs)
* [MethylSeekR](http://www.bioconductor.org/packages/release/bioc/html/MethylSeekR.html)
* Annotation of DMRs
* [genomation](https://bioconductor.org/packages/release/bioc/html/genomation.html)
* [ChIPpeakAnno](https://www.bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html)
* Web-based service
* [WBSA](http://wbsa.big.ac.cn/)
* **IP data**:
* Overview to CHIP-Seq:
* peak calling: [MACS2](https://github.com/taoliu/MACS/wiki/Advanced:-Call-peaks-using-MACS2-subcommands)
* Peak annotation and visualization
* [HOMER annotatePeak](http://homer.ucsd.edu/homer/ngs/annotation.html)
* [ChIPseeker](http://bioconductor.org/packages/release/bioc/html/ChIPseeker.html)
* Gene set enrichment analysis for ChIP-seq peaks
* [GREAT](http://bejerano.stanford.edu/great/public/html/)
### **(6.2) DNAase-seq**
* review : [Yongjing Liu, et al. Brief in Bioinformatics, 2019.](https://academic.oup.com/bib/article-abstract/20/5/1865/5053117?redirectedFrom=fulltext)
* Peak calling: [F-Seq](http://fureylab.web.unc.edu/software/fseq/)
* Peak annotation: [ChIPpeakAnno](https://www.bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html)
* Motif analysis: [MEME-ChIP](http://meme-suite.org/doc/meme-chip.html?man_type=web)
### **(6.3) ATAC-seq**
* Pipeline recommended by [Harward informatics](https://github.com/harvardinformatics/ATAC-seq)
### (7) Microbe data analysis
* [kraken2](https://ccb.jhu.edu/software/kraken2/): k-mer based fast metagenome reads classification
* [metaphlan](https://huttenhower.sph.harvard.edu/metaphlan/): marker gene based microbe taxonomy abundance estimation
* [motu](https://motu-tool.org/): marker gene based microbe taxonomy abundance estimation
* [maxbin](https://sourceforge.net/projects/maxbin/): binning contigs into metagenome-assembled genomes (MAGs)
* [mash](https://mash.readthedocs.io/en/latest/distances.html): rapid estimation of distance between genome
* [drep](https://drep.readthedocs.io/en/latest/): pick representative genome from sample-wise assembly
* [prodigal](https://github.com/hyattpd/Prodigal): prokaryote gene prediction
* [prokka](https://github.com/tseemann/prokka): pipeline for prokaryote genome annotation
* [qiime2](https://qiime2.org/): 16S amplicon sequencing data analysis
## More: Shared tools and scripts
* Scripts: [Lu Lab](https://github.com/lulab/shared_scripts) | [Zhi J. Lu](https://github.com/urluzhi/scripts)
* Plots: [Lu Lab](/teaching/part-i.-programming-skills/2.r/2.2.plots-with-r.md) | [Zhi J. Lu](https://github.com/urluzhi/scripts/tree/master/Rscript/R_plot)
## More: Software for the ages
| Software | Purpose | Creators | Key capabilities | Year released | Citationsa |
| ------------------------------------------- | ------------------------------------ | -------------------------------------------------------------------- | ------------------------------------------------------------------------------------------------ | :-----------: | :--------: |
| BLAST | Sequence alignment | Stephen Altschul, Warren Gish, Gene Myers, Webb Miller, David Lipman | First program to provide statistics for sequence alignment, combination of sensitivity and speed | 1990 | 35,617 |
| R | Statistical analyses | Robert Gentleman, Ross Ihaka | Interactive statistical analysis, extendable by packages | 1996 | N/A |
| ImageJ | Image analysis | Wayne Rasband | Flexibility and extensibility | 1997 | N/A |
| Cytoscape | Network visualization and analysis | Trey Ideker *et al*. | Extendable by plugins | 2003 | 2,374 |
| Bioconductor | Analysis of genomic data | Robert Gentleman *et al*. | Built on R, provides tools to enhance reproducibility of research | 2004 | 3,517 |
| Galaxy | Web-based analysis platform | Anton Nekrutenko, James Taylor | Provides easy access to high-performance computing | 2005 | 309b |
| MAQ | Short-read mapping | Heng Li, Richard Durbin | Integrated read mapping and SNP calling, introduced mapping quality scores | 2008 | 1,027 |
| Bowtie | Short-read mapping | Ben Langmead, Cole Trapnell, Mihai Pop, Steven Salzberg | Fast alignment allowing gaps and mismatches based on Burrows-Wheeler Transform | 2009 | 1,871 |
| Tophat | RNA-seq read mapping | Cole Trapnell, Lior Pachter, Steven Salzberg | Discovery of novel splice sites | 2009 | 817 |
| BWA | Short-read mapping | Heng Li, Richard Durbin | Fast alignment allowing gaps and mismatches based on Burrows-Wheeler Transform | 2009 | 1,556 |
| Circos | Data visualization | Martin Krzywinski *et al*. | Compact representation of similarities and differences arising from comparison between genomes | 2009 | 431 |
| SAMtools | Short-read data format and utilities | Heng Li, Richard Durbin | Storage of large nucleotide sequence alignments | 2009 | 1,551 |
| Cufflinks | RNA-seq analysis | Cole Trapnell, Steven Salzberg, Barbara Wold, Lior Pachter | Transcript assembly and quantification | 2010 | 710 |
| IGV | Short-read data visualization | James Robinson *et al*. | Scalability, real-time data exploration | 2011 | 335 |
| N/A, paper not available in Web of Science. | | | | | |
> From: [The anatomy of successful computational biology software](https://www.nature.com/articles/nbt.2721)
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