RNA-seq 분석 파이프라인 - 양복 맞춤에서 메타포를 따오다.

분석 절차 #

1. Quality control
   • FastQC
   • cutadapt or Trimmomatic: trimming adapter
   • FastQ Screen: check the composition of library sequences
2. Mapping
   • Bowtie
   • TopHat
   • STAR
3. Gene expression quantification
   • Cufflinks
   • featureCounts and TCC

4. Differential expression analysis

   • edgeR
   • DESeq
   • CummeRbund

5. GSEA

정량하는 단위 #

• RPKM: Reads per kilobase per million mapped reads - 라이브러리 크기와 길이로 나눠줌
• FPKM: Fragments per kilobase of model per million mapped fragments
• TPM: Transcripts per million

다양한 분석 파이프라인 #

Basic: TopHat - Cufflinks - CummeRbund #

TopHat - Cufflinks - CummeRbund

STAR - Cufflinks #

기본 방법에서 TopHat 대신 STAR를 사용함

Trimmomatic --> STAR --> Cufflinks

# Trimmomatic
$ java -jar trimmomatic-0.36.jar PE -phred33 read.R1.fq.gz read.R2.fq.gz read.R1.fq.gz read.R1.unpaired.fa.qz read.R2.fq.gz read.R2.unpaired.fq.gz ILLUMINACLIP:Trimmomatic-0.36/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

# STAR genome indexing
$ STAR --runThreadN 12 --runMode genomeGenerate --genomeDir genome --genomeFastaFiles Homo_sapiens.GRCh38.dna.primary_assembly.fa

# STAR mapping and 
$ STAR --runThreadN 12 --sjdbGTFfile Homo_sapiens.GRCh38.79.gtf --sjdbOverhang 100 --readFilesIn read.R1.fq.gz read.R2.fq.gz --readFilesCommand zcat --genomeDir genome

# sam to sorted bam
$ samtools view -bS Aligned.out.sam -o Aligned.out.bam
$ samtools sort Aligned.out.bam -o Aligned.out.sorted.bam

# Cufflinks
$ cufflinks -p 8 -o output Aligned.out.sorted.bam

HISAT - StringTie - Ballgown #

• Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown Nature Protocols 2016-08-11: HISAT, StringTie, Ballgown을 이용한 방법

기타 방법들 #

hppRNA #

• hppRNA-a Snakemake-based handy parameter-free pipeline for RNA-Seq analysis of numerous samples Briefings in Bioinformatics 2017-01-17: Snakemake 기반의 다양한 파이프라인 자동화

VIPER #

• VIPER: Visualization Pipeline for RNA-seq, a Snakemake workflow for efficient and complete RNA-seq analysis BMC Bioinformatics 2018-04-12: VIPER - Snakemake로 풀 자동화 - HTML 보고서까지

BioJupies #

• BioJupies: Automated Generation of Interactive Notebooks for RNA-seq Data Analysis in the Cloud bioRxiv 2018-06-20: 온라인에서 자동 분석, GEO 데이터도 분석 후 결과를 Jupyter notebook으로 내보냄

관련정보 #

• Galaxy RNA-seq analysis: Tuxedo protocol 2013-12-28: Galaxy를 이용하여 분석하기
• Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks 2012-03-01

Incoming Links #

Related Medical Scholarly Articles (MedicalScholarlyArticle 0) #

• Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown

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