• 向SRA提交数据 SRA Submission
• 知乎搜索 https://www.zhihu.com/search?q=rnaseq%20fastq%20star&type=content
• RNA-Seq数据标准化方法 https://zhuanlan.zhihu.com/p/37196518
• RNA-seq 保姆教程：差异表达分析（一） https://zhuanlan.zhihu.com/p/585176027
• RNA-seq：转录组数据分析处理(上) https://zhuanlan.zhihu.com/p/61847802
• Common File Formats Used by the ENCODE Consortium https://www.encodeproject.org/help/file-formats/
• FAA RNA Seq Compare https://www.faa.gov/sites/faa.gov/files/GEN_21001B_diffExp_TechReport.pdf

https://bioconductor.org/packages/release/BiocViews.html#___GeneExpressionWorkflow
Raw Data: Fastq (.gz) →

1. QC: FastQC
2. Filter
3. Alignment: STAR
4. Count
5. Cluster
6. Heatmap
7. Differential

apt install fastqc

fastqc --noextract RawData/I409/I409_1.fq.gz -o results/1_initial_qc/ 

• Ensembl: https://ensemblgenomes.org/ https://useast.ensembl.org/index.html
• FTP: https://ftp.ensembl.org/pub/rapid-release/

Rat https://useast.ensembl.org/Rattus_norvegicus/Info/Index

GTF https://ftp.ensembl.org/pub/release-109/gtf/rattus_norvegicus/
GFF https://ftp.ensembl.org/pub/release-109/gff3/rattus_norvegicus/

https://registry.opendata.aws/jhu-indexes/

• https://github.com/pachterlab/kallisto
• STAR
• HISAT2
• Salmon https://combine-lab.github.io/salmon/getting_started/

https://daehwankimlab.github.io/hisat2/download/ * https://notebook.community/ssjunnebo/pathogen-informatics-training/Notebooks/RNA-Seq/genome-mapping

hisat2-build -p 32 fasta/Rattus_norvegicus.mRatBN7.2.dna.toplevel.fa hisat_index
hisat2 -x hisat_index/hisat_index -1 I409/I409_1.fq.gz -2 I409/I409_2.fq.gz -S I409.sam -p 32

2.7.10b

• Github:https://github.com/alexdobin/STAR
• Ubuntu Package:https://ubuntu.pkgs.org/22.04/ubuntu-universe-arm64/rna-star_2.7.10a+dfsg-1_arm64.deb.html

apt install rna-star

# 创建索引，索引文件创建一次即可. 需要从Ensembl下载对应物种的Fasta文件和GTF文件。
STAR --runMode genomeGenerate --genomeDir star_index --genomeFastaFiles fasta/* --sjdbGTFfile gtf/* --runThreadN 14

# 运行分析
STAR --genomeDir star_index --readFilesIn filtered/sample_filtered.fq --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --runThreadN 14

STAR --genomeDir star_index --readFilesIn rna4/RawData/I409/I409_1.fq --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --runThreadN 14

# bat
kallisto bus [arguments] FASTQ-files

kallisto quant -i rattus_index_ki/transcriptome.idx -o reads.kallisto_quant -t 64 --fusion --pseudobam --genomebam --gtf gtf\Rattus_norvegicus.mRatBN7.2.109.gtf rna4\RawData\I409\I409_1.fq.gz rna4\RawData\I409\I409_2.fq.gz

可以Reads的工具https://hbctraining.github.io/Intro-to-rnaseq-hpc-O2/lessons/05_counting_reads.html

1 https://subread.sourceforge.net/

featureCounts https://rnnh.github.io/bioinfo-notebook/docs/featureCounts.html

featureCounts -p -M -O -T 32 -a gtf/Rattus_norvegicus.mRatBN7.2.109.gtf -o output.txt data.sam [bam]