写在前面

最近接二连三带了几个实习生和轮转生，每带一个人感觉总要说很多重复内容。因为可以预见后面几年还会再带不少人，想了想还是把一些东西写下来后面就可以直接丢链接。

本文列举生物信息部分常用工具和几个神奇网站，基本上每个工具都给出（或中文或英文）简要功能介绍和官网地址。

生物信息学常用工具

fastq格式相关

• SRAtoolkit

• 网址 https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc

• SRA数据库下载公用数据时的工具

• fastx toolkit

• a collection of command line tools for Short-Reads FASTA/FASTQ files preprocessing

• 有各种各样的小功能，比如提取反向互补序列等等。

• 网址 http://hannonlab.cshl.edu/fastx_toolkit/

• fastqc

• A quality control tool for high throughput sequence data

• 评估测序数据质量

• 网址 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

• MultQC

• Aggregate results from bioinformatics analyses across many samples into a single report

• 一次同时生成多个数据质量报告，省时省力方便对比，支持fastqc

• 网址 https://github.com/ewels/MultiQC

• 网址 http://multiqc.info/docs/

• Trim Galore

• around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files,

• with some extra functionality for MspI-digested RRBS-type (Reduced Representation Bisufite-Seq) libraries

• 和fastqc出自一家，可以和fastqc结合使用，用来清洗原始数据。

• 网址 https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/

• Trimmomatic

• A flexible read trimming tool for Illumina NGS data

• 专门清洗illumina测序数据的工具

• 网址 http://www.usadellab.org/cms/index.php?page=trimmomatic

• khmer

• working with DNA shotgun sequencing data from genomes, transcriptomes, metagenomes, and single cells.

• 可以对原始测序数据进行过滤等

• 网址 http://khmer.readthedocs.io/en/v2.1.1/user/scripts.htm

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BED格式相关

• bedops

• the fast, highly scalable and easily-parallelizable genome analysis toolkit

• 网址 https://bedops.readthedocs.io/en/latest/index.html

• 玩转bed格式文件，速度比bedtools快

• bedtools

• a powerful toolset for genome arithmetic

• 网址 http://bedtools.readthedocs.io/en/latest/index.html

• 最知名的bed文件相关工具，但是和samtools并非出自一家

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SAM/BAM格式相关

• samtools

• Utilities for the Sequence Alignment/Map (SAM) format

• 网址 http://www.htslib.org/doc/samtools.html

• 有这一个就够了

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SNP（VCF/BCF）相关

• GATK

• 网址 https://software.broadinstitute.org/gatk/documentation/

• 使用率最高的软件

• bcftools

• utilities for variant calling and manipulating VCFs and BCFs

• 网址 http://www.htslib.org/doc/bcftools.html

• 对vcf格式的文件进行各种操作

• vcftools

• 网址 https://vcftools.github.io/man_latest.html

• 和bcftools类似

• snpEFF

• Genetic variant annotation and effect prediction toolbox

• 适合用来进行snp注释

• 用法 http://snpeff.sourceforge.net/SnpEff_manual.html

• 网址 http://snpeff.sourceforge.net/

• 也可以注释ChIP-seq

• 支持非编码注释，如组蛋白修饰

• samtools mpileup

• Utilities for the Sequence Alignment/Map (SAM) format

• 网址 http://www.htslib.org/doc/samtools.html

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ChIP-seq/motif

• MACS

• Model-based Analysis of ChIP-Seq

• 主要用于组蛋白修饰产生的narrow peaks(H3K4me3 and H3K9/27ac)

• transcription factors which are usually associated with sharp and solated peaks

• 网址 http://liulab.dfci.harvard.edu/MACS/README.html

• MACS2

• 网址 https://github.com/taoliu/MACS

• MACS的升级版本，也可以用来找broad peak

• SICER

• highly recommended for a practical ChIP-seq experiment design and can be used to account for local biases resulting from read mappability, DNA repeats, local GC content

• 网址https://www.genomatix.de/online_help/help_regionminer/sicer.html

• 出来怼MACS，主要用来找一些比较宽的peak,类似于H3K9me3 和 H3K36me3。

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large sequences alignment

长序列比对常用的几个软件

• MUMer

• rapid alignment of very large DNA and amino acid sequences

• 网址 http://mummer.sourceforge.net/examples/

• 网址 http://mummer.sourceforge.net/manual/

• GMAP

• GMAP: A Genomic Mapping and Alignment Program for mRNA and EST Sequences

• 网址 http://research-pub.gene.com/gmap/

• BLAT

• Blat produces two major classes of alignments:at the DNA level between two sequences that are of 95% or greater identity, but which may include large inserts；at the protein or translated DNA level between sequences that are of 80% or greater identity and may also include large inserts.

• 网址 https://genome.ucsc.edu/goldenpath/help/blatSpec.html

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short reads alignment

短序列比对，二代测序数据比对

• BWA

• Burrows-Wheeler Alignment Tool

• mapping low-divergent sequences against a large reference genome

• It consists of three algorithms: BWA-backtrack, BWA-SW and BWA-MEM.

• 网址 http://bio-bwa.sourceforge.net/bwa.shtml

• 网址 https://github.com/lh3/bwa

• GSNAP

• Genomic Short-read Nucleotide Alignment Program

• 网址 http://research-pub.gene.com/gmap/

• Bowtie

• works best when aligning short reads to large genomes

• not yet report gapped alignments

• 网址 http://bowtie-bio.sourceforge.net/manual.shtml

• Bowtie2

• ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences

• supports gapped, local, and paired-end alignment modes

• 网址 http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#reporting

• 和上一代的区别在于支持gapped alignments

• HISAT2

• Tophat的继任者，基于HISAT和Bowtie2

• HISAT2的速度比STAR快一些

• 网址 http://ccb.jhu.edu/software/hisat2/manual.shtml

• STAR

• Spliced Transcripts Alignment to a Reference

• 网址 https://github.com/alexdobin/STAR

• 网址 https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf

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genome guide assemble

• stringtie

• highly efficient assembler of RNA-Seq alignments into potential transcripts

• 对于可变剪切的发现相对准确

• 网址 https://ccb.jhu.edu/software/stringtie/

• Cufflinks

• 基本不用了。。。

• IDP

• Isoform Detection and Prediction tool

• gmap+hisat2,也就是长短序列比对相结合，效果不错

• 网址 https://www.healthcare.uiowa.edu/labs/au/IDP/IDP_manual.asp

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de novo assemble/gene prediction

下面几个软件结合起来就是一个从组装到注释到计算效率的过程

• trintiy

• 倾向于预测长的可变剪接

• 新版本从之前的过度预测越来越倾向于有所保留

• 比较耗资源，一般1个CPU最好分配6G-10G

• 可以有参或者无参转录组拼接

• 网址 https://github.com/trinityrnaseq/trinityrnaseq/wiki

• oases

• 通常得到的N50比较高

• 检测低表达的基因有一定优势

• De novo transcriptome assembler for very short reads

• 网址 https://github.com/dzerbino/oases

• PASA（内包括BLAT和GMAP）

• 得到拼接好的fasta文件后可以用pasa进行基因结构预测

• Gene Structure Annotation and Analysis Using PASA

• 网址 http://pasapipeline.github.io/

• Maker

• 基因预测

• can be used for de novo annotation of newly sequenced genomes, for updating existing annotations to reflect new evidence, or just to combine annotations, evidence, and quality control statistics

• 网址 http://www.yandell-lab.org/software/maker.html

• TransRate

• reference free quality assessment of de novo transcriptome assemblies

• 网址 http://hibberdlab.com/transrate/

• 专业的拼接质量评估软件，有三种评估模式。

• DETONATE

• DE novo TranscriptOme rNa-seq Assembly with or without the Truth Evaluation

• 网址 https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0553-5

• BUSCO

• 它的评估模式和上面两个不太一样

• based on evolutionarily-informed expectations of gene content from near-universal single-copy orthologs

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Estimating transcript abundance

可以分为基于比对和不基于比对两种，其中RSEM和eXpress是基于比对的，另外两种是基于比对的。

• RSEM

• RNA-Seq by Expectation-Maximization

• 网址 https://deweylab.github.io/RSEM/README.html

• eXpress

• quantifying the abundances of a set of target sequences from sampled subsequences

• 网址 https://pachterlab.github.io/eXpress/overview.html

• kallisto

• 快到飞起

• 丰度估计中样本特异性和读长偏好性低

• quantifying abundances of transcripts from RNA-Seq data

• 网址 https://pachterlab.github.io/kallisto/

• salmon

• 网址 quantifying the expression of transcripts using RNA-seq data

• https://combine-lab.github.io/salmon/

• 也是很快

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Read count

• htseq-count

• 网址 http://htseq.readthedocs.io/en/release_0.9.1/

• 数read, 有它就够了

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Difference expression

和之前的步骤对应，这里也可以分为基于read和基于组装以及不基于比对三类工具。

• limma

• Linear Models for Microarray Data

• 网址 http://bioconductor.org/packages/release/bioc/html/limma.html

• 用于分析芯片数据

• DEseq

• 网址 http://bioconductor.org/packages/release/bioc/html/DESeq.html

• DEseq2

• 效果在几个工具中相对好

• 网址 http://bioconductor.org/packages/release/bioc/html/DESeq2.html

• DEGseq

• Identify Differentially Expressed Genes from RNA-seq data

• 网址 http://www.bioconductor.org/packages/2.6/bioc/html/DEGseq.html

• edgeR

• Empirical Analysis of Digital Gene Expression Data in R

• 网址 http://www.bioconductor.org/packages/release/bioc/html/edgeR.html

• Ballgown

• 准确度有时不是很好

• facilitate flexible differential expression analysis of RNA-Seq data

• organize, visualize, and analyze the expression measurements for your transcriptome assembly.

• 网址 https://github.com/alyssafrazee/ballgown

• sleuth

• 用来配合kallisto使用

• 网址 https://pachterlab.github.io/sleuth/about

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Data visualization

数据可视化的工具可以分为本地版本和在线版本

• IGV

• Integrative Genomics Viewer

• 网址 http://software.broadinstitute.org/software/igv/

• 本地展示分析结果的不二选择

• jbrowse

• 公开展示数据或者给合作者分享时的不二选择，快且好看。

• 网址 http://jbrowse.org/code/JBrowse-1.10.2/docs/tutorial/

• DEIVA

• Interactive Visual Analysis of differential gene expression test results

• 网址 http://hypercubed.github.io/DEIVA/

• 差异表达的可视化在线工具

• Heatmapper

• expression-based heat maps

• pairwise distance maps

• correlation maps

• 网址 http://www.heatmapper.ca/

• 用来话各种热图的在线工具

• START

• visualize RNA-seq data starting with count data

• 网址 https://kcvi.shinyapps.io/START/

• 基于shinny的一套RNA-seq数据可视化工具

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几个神奇的网站

• biostars

• 网址 https://www.biostars.org

• R book

• 网址 http://r4ds.had.co.nz/

• python guide

• 网址 http://docs.python-guide.org/en/latest/

• bioptyhon

• 网址 http://biopython.org/DIST/docs/tutorial/Tutorial.html

• Rosalind

• 网址 http://rosalind.info/problems/list-view/

• bioinformatics tools

• 网址 https://omictools.com/

• 网址 https://bioinformatics.ca/links_directory/

• data visualistion catalogue

• 网址 http://datavizcatalogue.com/index.html

暂时就写这么多，还有一些自己平时也很少用的就不放进来给他人增加负担，后面再进行补充。

当然，我怎么会告诉你 阅读原文 可以直接点击文中出现的各种链接呢？

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