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====== RNA-Seq ======
====教程====
  * 向SRA提交数据 [[stat:rnaseq:srasubmit]]
  * 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]]

=====流程=====
[[https://bioconductor.org/packages/release/BiocViews.html#___GeneExpressionWorkflow]]\\
Raw Data: Fastq (.gz) ->
  - QC: FastQC
  - Filter
  - Alignment: STAR 
  - Count
  - Cluster
  - Heatmap
  - Differential

====1 QC ====
===FastQC===
<code>
apt install fastqc

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


====2 Alignment====

===基因组注释数据===
  * 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/]]

===Alignment Indexing===
[[https://registry.opendata.aws/jhu-indexes/]]


===对齐工具===
  * [[https://github.com/pachterlab/kallisto]]
  * STAR
  * HISAT2
  * Salmon [[https://combine-lab.github.io/salmon/getting_started/]]

===HISAT2===
[[https://daehwankimlab.github.io/hisat2/download/]]
 *  [[https://notebook.community/ssjunnebo/pathogen-informatics-training/Notebooks/RNA-Seq/genome-mapping]]

<code>
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

</code>

===STAR===
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]]
<code bash>
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
</code>

=== kallisto  ===
<code>
# 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

</code>
====3 Count ====
>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]]
<code>
featureCounts -p -M -O -T 32 -a gtf/Rattus_norvegicus.mRatBN7.2.109.gtf -o output.txt data.sam [bam]
</code>
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