史上最全 39个RNAseq分析工具与对比

转录组分析工具超强整理！文末有惊喜哦~...

文献：Sahraeian S M E, Mohiyuddin M, Sebra R, et al. Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis[J]. Nature Communications, 2017, 8(1):59.

这是一篇在NC上发表的使用RNAseq工具对比的一篇文献，解读这篇文献对我们使用RNAseq发文提供了思路。下面小编具体解说一下。

文献摘要：

RNA-sequencing(RNA-seq)是一个重要的转录组学研究技术,数百款分析工具目前已经开发出来。尽管最近相关研究评估了最新的可用的RNAseq工具,但他们没有全面综合的评估RNAseq分析的工作流。这里我们进行广泛的RNA-seq工作流的研究分析，不仅包括表达分析,我们的工作还包括了评估的RNA variant-calling,RNA编辑和RNA融合检测技术。更为独特的是我们对二代RNAseq和三代Isoseq技术都进行了研究,39个分析工具，~ 120种组合,涉及15个样品与各种生殖系、癌症和干细胞的数据集的~490种分析。我们报告了各流程性能并提出一个全面的，分析准确性高的RNA-seq分析流程,名字叫做RNACocktail。在不同的样品中验证表明,我们提出的流程可以帮助研究人员通过转录组的分析获取更多的生物有关的预测结果。

流程下载地址：http://bioinform.github.io/rnacocktail/

附录：39个工具版本号、重要参数及下载地址：

比对工具

1.TopHat2： –no-coverage-search

http://ccb.jhu.edu/software/tophat/index.shtml

2.STAR: -twopassMode Basic –outFilterType BySJout

https://github.com/alexdobin/STAR/releases

3.HISAT2 2.0.1-beta –dta (or –dta-cufflinks)

http://www.ccb.jhu.edu/software/hisat/index.shtml

4.RASER 0.52 -b 0.03

https://www.ibp.ucla.edu/research/xiao/RASER.html

有参考转录本组装工具

1.Cufflinks 2.2.1 –frag-bias-correct

http://cole-trapnell-lab.github.io/cufflinks/

2.StringTie 1.2.1 -v -B

http://www.ccb.jhu.edu/software/stringtie/

无参考转录本组装工具

1.SOAPdenovoTrans 1.04 -K 25

https://github.com/aquaskyline/SOAPdenovo-Trans/

2.Oases 0.2.09 (Velvetv1.2.10) (velveth haslength: 25) (velvetg options: -read trkg yes)

http://www.ebi.ac.uk/~zerbino/oases/

3. Trinity 2.1.1 –normalize reads

http://trinityrnaseq.sourceforge.net/

三代长read分析工具

1.LoRDEC 0.6 -k 23 -s 3

http://atgc.lirmm.fr/lordec/

2.GMAP 12/31/15 -f 1

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

3. STARlong 2.5.1b

https://github.com/alexdobin/STAR/releases

Followed the recommended options :

–outSAMattributes NH HI NM MD

–readNameSeparator space

–outFilterMultimapScoreRange 1

–outFilterMismatchNmax 2000

–scoreGapNoncan -20

–scoreGapGCAG -4

–scoreGapATAC -8

–scoreDelOpen -1

–scoreDelBase -1

–scoreInsOpen -1

–scoreInsBase -1

–alignEndsType Local

–seedSearchStartLmax 50

–seedPerReadNmax 100000

–seedPerWindowNmax 1000

–alignTranscriptsPerReadNmax 100000

–alignTranscriptsPerWindowNmax 10000

–outSAMstrandField intronMotif

–outSAMunmapped Within

4. IDP 0.1.9

https://www.healthcare.uiowa.edu/labs/au/IDP/

定量工具

1. eXpress 1.5.1 (bowtie2 v2.2.7) (bowtie2 options: -a -X 600 –rdg 6,5 –rfg 6,5 –score-min L,-.6,-.4 –no-discordant –no-mixed)

https://pachterlab.github.io/eXpress/index.html

2. kallisto 0.42.4

http://pachterlab.github.io/kallisto/about.html

3. Sailfish 0.9.0

http://www.cs.cmu.edu/~ckingsf/software/sailfish/

4. Salmon-Aln 0.6.1

https://github.com/COMBINE-lab/salmon

5. Salmon-SMEM 0.6.1

https://github.com/COMBINE-lab/salmon

index: –type fmd

quant: -k,19

6. Salmon-Quasi 0.6.1

https://github.com/COMBINE-lab/salmon

index: –type quasi -k 31

7. featureCounts 1.5.0-p1 -p -B -C

http://subread.sourceforge.net/

差异表达分析工具

1. DESeq2 1.14.1

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

2. edgeR 3.16.5

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

3. limma 3.30.7

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

4. Cuffdiff 2.2.1

–frag-bias-correct –emit-count-tables

http://cole-trapnell-lab.github.io/cufflinks/

5. Ballgown 2.6.0

https://github.com/alyssafrazee/ballgown

6. sleuth 0.28.1

https://github.com/pachterlab/sleuth

变异分析工具

1. SAMtools 1.2 (bcftools v1.2)

samtools mpileup -C50 -d 100000

https://github.com/samtools/samtools

2. bcftools filter -s LowQual -e ‘%QUAL10000’

https://github.com/samtools/bcftools

3.GATK v3.5-0-g36282e4 (picard 1.129)

https://software.broadinstitute.org/gatk/download/

Picard AddOrReplaceReadGroups: SO=coordinate

Picard MarkDuplicates: CREATE INDEX=true VALIDATION STRINGENCY=SILENTGATK

SplitNCigarReads: -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60

-U ALLOW N CIGAR READSGATK

HaplotypeCaller: -stand call conf 20.0

-stand emit conf 20.0 -A StrandBiasBySample

-A StrandAlleleCountsBySampleGATK

VariantFiltration: -window 35 -cluster 3 -filterName FS -filter

“FS >30.0” -filterName QD -filter “QD

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