RiboParser 0.1.3__tar.gz

riboparser-0.1.3/PKG-INFO

@@ -0,0 +1,1316 @@
+Metadata-Version: 2.1
+Name: RiboParser
+Version: 0.1.3
+Summary: A pipeline for ribosome profiling data analysis
+Home-page: https://github.com/renscq/RiboParser
+Author: Ren Shuchao
+Author-email: rensc0718@163.com
+Classifier: Programming Language :: Python :: 3
+Classifier: License :: OSI Approved :: MIT License
+Classifier: Operating System :: OS Independent
+Requires-Python: >=3.12
+Description-Content-Type: text/markdown
+Requires-Dist: numpy~=1.26.4
+Requires-Dist: pandas~=2.2.2
+Requires-Dist: pyarrow~=16.1.0
+Requires-Dist: polars~=0.20.31
+Requires-Dist: biopython~=1.78
+Requires-Dist: scipy~=1.12.0
+Requires-Dist: scikit-learn~=1.4.2
+Requires-Dist: statsmodels~=0.14.2
+Requires-Dist: pysam~=0.22.1
+Requires-Dist: joblib~=1.4.2
+Requires-Dist: interval~=1.0.0
+Requires-Dist: matplotlib~=3.8.4
+Requires-Dist: matplotlib-venn~=1.1.1
+Requires-Dist: seaborn~=0.13.2
+Requires-Dist: plotly~=5.22.0
+Requires-Dist: seqlogo~=5.29.9
+Requires-Dist: kaleido~=0.2.1
+
+<!--
+ * @Author: 'rensc' 'rensc0718@163.com'
+ * @Date: 2024-10-15 11:44:58
+ * @LastEditors: 'rensc' 'rensc0718@163.com'
+ * @LastEditTime: 2024-10-20 07:06:36
+ * @FilePath: \RiboParser\README.md 
+ * 
+-->
+
+# RiboParser
+
+为了便于理解和使用，这里对公开的项目数据进行分析，并拆解每一个分析步骤，来展示完整的工作流程。
+这个过程包括了通用的分析步骤，以及定制的 `RiboParser` 和 `RiboShiny` 的分析和可视化步骤。
+
+1. 软件的安装
+2. 参考文件的创建
+3. 原始数据的下载
+4. 原始数据清洗
+5. 数据比对
+6. 测序质量分析
+7. 基因水平分析
+8. 密码子水平分析
+
+以上的数据分析输出的结果可以在 `RiboShiny` 中进行下游的分析和可视化。
+
+
+## 1. 软件的安装
+
+### 1. conda 创建环境
+```bash
+conda create -n ribo
+conda activate ribo
+```
+
+### 2. conda 安装软件依赖
+```bash
+conda install cutadapt
+conda install bowtie
+conda install samtools
+conda install star
+conda install bedtools
+conda install subread
+conda install rsem
+conda install pigz
+conda install gffread
+conda install sra-tools
+conda install ucsc-genepredtogtf
+conda install ucsc-gtftogenepred
+conda install ucsc-gff3togenepred
+conda install ucsc-bedgraphtobigwig
+conda install ucsc-bedsort
+```
+
+### 3. conda 安装 RiboParser
+```bash
+conda install riboparser -c rensc
+```
+
+### 4. 测试安装状态：
+测试软件的依赖、安装和运行问题。
+
+```bash
+rpf_test
+```
+
+## 2. 准备参考文件
+
+### 1. 完整项目目录示例如下：
+
+完整的数据分析包含了参考文献的准备、RNA-seq的数据分析、Ribo-seq 的数据分析。
+
+```
+$ cd /mnt/t64/test/sce/
+$ tree
+
+.
+├── 1.reference
+│   ├── cdna
+│   ├── genome
+│   ├── gtf
+│   ├── mrna
+│   ├── norm
+│   ├── ncrna
+│   ├── rrna
+│   ├── rsem-index
+│   ├── star-index
+│   └── trna
+├── 2.rawdata
+│   ├── rna-seq
+│   └── ribo-seq
+├── 3.rna-seq
+│   ├── 1.cleandata
+│   ├── 2.bowtie
+│   ├── 3.star
+│   ├── 4.quantification
+│   └── 5.riboparser
+│       ├── 01.qc
+│       ├── 03.offset
+│       ├── 04.density
+│       ├── 05.merge
+│       ├── 06.periodicity
+│       ├── 07.metaplot
+│       ├── 08.coverage
+│       ├── 09.correlation
+│       ├── 10.shuffle
+│       └── 11.gene_density
+├── 4.ribo-seq
+│   ├── 1.cleandata
+│   ├── 2.bowtie
+│   ├── 3.star
+│   ├── 4.quantification
+│   └── 5.riboparser
+│       ├── 01.qc
+│       ├── 02.digestion
+│       ├── 03.offset
+│       ├── 04.density
+│       ├── 05.merge
+│       ├── 06.periodicity
+│       ├── 07.metaplot
+│       ├── 08.coverage
+│       ├── 09.correlation
+│       ├── 10.quantification
+│       ├── 11.pausing_score
+│       ├── 12.codon_occupancy
+│       ├── 13.codon_decoding_time
+│       ├── 14.codon_selection_time
+│       ├── 15.coefficient_of_variation
+│       ├── 16.meta_codon
+│       ├── 17.shuffle
+│       └── 18.gene_density
+└── 5.test
+
+```
+
+### 2. 准本参考基因组索引
+
+#### 2.1. 创建目录
+
+创建文件夹用于放置不同类型的参考序列文件。
+
+```bash
+$ cd /mnt/t64/test/sce/1.reference/
+
+$ mkdir cdna genome gtf mrna ncrna rrna trna norm rsem-index
+```
+
+#### 2.2 从 NCBI 下载参考文件
+
+使用最常用的数据分析文件格式，基因组序列为 fasta 格式，参考文件为 GTF 或者 GFF3 格式。
+
+```bash
+# genome sequence
+$ wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.fna.gz
+
+# GTF or GFF3
+$ wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.gtf.gz
+$ wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.gff.gz
+
+# cDNA sequence
+$ wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_rna.fna.gz
+
+# feature table
+$ wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_feature_table.txt.gz
+
+# decompression
+$ gunzip *.gz
+
+$ gffread -g GCF_000146045.2_R64_genomic.fna GCF_000146045.2_R64_genomic.gff -F -w cdna.fa
+```
+
+#### 2.3 使用 bowtie 创建 genome 索引
+
+```bash
+$ cd /mnt/t64/test/sce/1.reference/genome
+
+$ bowtie-build ../GCF_000146045.2_R64_genomic.fna genome.fa genome
+```
+
+#### 2.4 使用 bowtie 创建 mRNA 索引
+
+```bash
+$ cd mrna
+
+# filter the mrna sequence
+$ grep -i 'gbkey=mRNA' cdna.fa | cut -d ' ' -f 1 | cut -c 2- > mrna.ids
+
+$ retrieve_seq -i cdna.fa -n mrna.ids -o mrna.fa
+
+$ bowtie-build mrna.fa mrna
+```
+
+#### 2.5 使用 bowtie 创建 rRNA 索引
+```bash
+$ cd /mnt/t64/test/sce/1.reference/rrna
+
+# filter the rrna sequence
+$ grep -i 'gbkey=rRNA' cdna.fa | cut -d ' ' -f 1 | cut -c 2- > rrna.ids
+
+$ retrieve_seq -i cdna.fa -n rrna.ids -o rrna.fa
+
+$ bowtie-build rrna.fa rrna
+```
+
+#### 2.6 使用 bowtie 创建 tRNA 索引
+```bash
+$ cd /mnt/t64/test/sce/1.reference/trna
+
+# filter the trna sequence
+$ grep -i 'gbkey=tRNA' cdna.fa | cut -d ' ' -f 1 | cut -c 2- > trna.ids
+
+$ retrieve_seq -i cdna.fa -n trna.ids -o trna.fa
+
+$ bowtie-build trna.fa trna
+```
+
+
+#### 2.7 使用 bowtie 创建 ncRNA 索引
+```bash
+$ cd /mnt/t64/test/sce/1.reference/ncrna
+
+# filter the ncrna sequence
+$ grep -iE 'gbkey=ncRNA|gbkey=lnc_RNA|gbkey=miRNA|gbkey=snoRNA|gbkey=snRNA|gbkey=misc_RNA' cdna.fa | cut -d ' ' -f 1 | cut -c 2- > ncrna.ids
+
+$ retrieve_seq -i cdna.fa -n ncrna.ids -o ncrna.fa
+
+$ bowtie-build ncrna.fa ncrna
+```
+
+#### 2.8 标准化 gtf 文件
+```bash
+$ cd /mnt/t64/test/sce/1.reference/norm/
+
+$ rpf_Reference \
+ -g ../GCF_000146045.2_R64_genomic.fna \
+ -t ../GCF_000146045.2_R64_genomic.gff \
+ -u 30 -o sce
+```
+
+#### 2.9 使用 star 创建 genome 索引
+```bash
+$ cd /mnt/t64/test/sce/1.reference/
+
+$ STAR \
+ --genomeSAindexNbases 11 \
+ --runThreadN 12 \
+ --runMode genomeGenerate \
+ --genomeDir star-index \
+ --genomeFastaFiles GCF_000146045.2_R64_genomic.fna \
+ --sjdbGTFfile ./norm/sce.norm.gtf
+
+```
+
+#### 2.10 使用 rsem 创建 transcriptome 索引
+```bash
+$ cd /mnt/t64/test/sce/1.reference/rsem-index/
+
+$ rsem-prepare-reference \
+ -p 10 \
+ --gtf ../norm/sce.norm.gtf ../GCF_000146045.2_R64_genomic.fna sce
+
+```
+
+
+## 3. 示例
+为了展示 RiboParser 的分析流程和使用方法，这里使用数据集 GSE67387 的 RNA-seq 和 Ribo-seq 数据做示例。
+
+```shell
+# dataset
+https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67387
+
+# reference
+Nedialkova DD, Leidel SA. Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity. Cell 2015 Jun 18;161(7):1606-18. 
+PMID: 26052047
+```
+
+
+### 3.1 GSE67387 数据基础分析
+
+#### 3.1.1 下载原始数据
+1. 下载 RNA-seq 数据
+使用 `sra-tools` 中的 `prefetch` 下载原始的 sra 格式数据，并解压为 fastq 格式文件。
+```bash
+$ cd /mnt/t64/test/sce/2.rawdata/rna-seq/
+
+#################################################
+# download rna-seq
+$ prefetch -o SRR1944925.sra SRR1944925
+$ prefetch -o SRR1944926.sra SRR1944926
+$ prefetch -o SRR1944927.sra SRR1944927
+$ prefetch -o SRR1944928.sra SRR1944928
+$ prefetch -o SRR1944929.sra SRR1944929
+$ prefetch -o SRR1944930.sra SRR1944930
+$ prefetch -o SRR1944931.sra SRR1944931
+$ prefetch -o SRR1944932.sra SRR1944932
+$ prefetch -o SRR1944933.sra SRR1944933
+$ prefetch -o SRR1944934.sra SRR1944934
+$ prefetch -o SRR1944935.sra SRR1944935
+
+# decompression
+for sra in *.sra
+do
+fastq-dump $sra
+pigz *fastq
+done
+```
+
+2. 下载 Ribo-seq 数据
+```bash
+cd /mnt/t64/test/sce/2.rawdata/ribo-seq/
+
+#################################################
+# download ribo-seq
+prefetch -o SRR1944912.sra SRR1944912
+prefetch -o SRR1944913.sra SRR1944913
+prefetch -o SRR1944914.sra SRR1944914
+prefetch -o SRR1944915.sra SRR1944915
+prefetch -o SRR1944916.sra SRR1944916
+prefetch -o SRR1944917.sra SRR1944917
+prefetch -o SRR1944918.sra SRR1944918
+prefetch -o SRR1944919.sra SRR1944919
+prefetch -o SRR1944920.sra SRR1944920
+prefetch -o SRR1944921.sra SRR1944921
+prefetch -o SRR1944922.sra SRR1944922
+prefetch -o SRR1944923.sra SRR1944923
+
+# decompression
+for sra in *.sra
+do
+fastq-dump $sra
+pigz *fastq
+done
+```
+
+
+#### 3.1.2 数据清洗
+因为该项目提供的原始数据是清洗后的，所以并不包含接头序列，这里只展示通用步骤。
+
+1. 清洗 RNA-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/1.cleandata/
+
+#################################################
+# run the cutadapt
+for fq in /mnt/t64/test/sce/2.rawdata/rna-seq/*fastq.gz
+do
+cutadapt --match-read-wildcards \
+ -a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC \
+ -m 10 -O 6 -j 10 \
+ -o `\basename $fq fastq.gz`clean.fastq.gz $fq &> $fq".log"
+done
+```
+
+2. 清洗 Ribo-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/1.cleandata/
+
+#################################################
+# run the cutadapt
+for fq in /mnt/t64/test/sce/2.rawdata/ribo-seq/*fastq.gz
+do
+cutadapt --match-read-wildcards \
+ -a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC \
+ -m 10 -O 6 -j 10 \
+ -o `\basename $fq fastq.gz`clean.fastq.gz $fq &> $fq".log"
+done
+```
+
+
+#### 3.1.3 把 clean data 比对到不同类型的参考文件
+为了确定文库的质量，排除不同 ncRNA 来源的 reads 对后续分析的影响，这里使用 `bowtie` 对数据进行分类。
+正常情况下，尤其是使用 oligoDT 方法构建的 RNA-seq 文库，其中的 reads 大多来源于 mRNA。所以对于 RNA-seq 的分析而言，这个步骤不是必须的。
+
+1. 比对 RNA-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/2.bowtie/
+
+#################################################
+# set database
+rrna='/mnt/t64/test/sce/1.reference/rrna/rrna'
+trna='/mnt/t64/test/sce/1.reference/trna/trna'
+ncrna='/mnt/t64/test/sce/1.reference/ncrna/ncrna'
+mrna='/mnt/t64/test/sce/1.reference/mrna/mrna'
+chrom='/mnt/t64/test/sce/1.reference/genome/genome'
+
+# alignment reads to reference
+for fq in /mnt/t64/test/sce/3.rna-seq/1.cleandata/*fastq.gz
+do
+fqname=`\basename $fq .fastq.gz`
+
+## rrna
+bowtie -p 10 -v 1 --un="$fqname".norrna.fq --al="$fqname".rrna.fq \
+ -x $rrna $fq -S "$fqname".rrna.sam 2>> "$fqname".log
+
+## trna
+bowtie -p 10 -v 1 --un="$fqname".notrna.fq --al="$fqname".trna.fq \
+ -x $trna "$fqname".norrna.fq -S "$fqname".trna.sam 2>> "$fqname".log
+
+## ncrna
+bowtie -p 10 -v 1 --un="$fqname".noncrna.fq --al="$fqname".ncrna.fq \
+ -x $ncrna "$fqname".notrna.fq -S "$fqname".ncrna.sam 2>> "$fqname".log
+
+## mrna
+bowtie -p 10 -v 1 --un="$fqname".nomrna.fq --al="$fqname".mrna.fq \
+ -x $mrna "$fqname".noncrna.fq -S "$fqname".mrna.sam 2>> "$fqname".log
+
+## genome
+bowtie -p 10 -v 1 --un="$fqname".nogenome.fq --al="$fqname".genome.fq 、
+ -x $chrom "$fqname".nomrna.fq -S "$fqname".genome.sam 2>> "$fqname".log
+
+## compress fastq
+pigz *fq
+
+## compress sam
+for sam in *.sam
+do
+samtools view -h -F 4 $sam | samtools sort -@ $threads -o `\basename $sam sam`bam
+rm $sam
+done
+
+done
+```
+
+2. 统计所有数据库的比对结果
+```bash
+#################################################
+# merge all log files
+merge_bwt_log \
+ -n rRNA,tRNA,ncRNA,mRNA,Genome \
+ -l *log -o sce
+
+```
+
+3. 比对 Ribo-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/2.bowtie/
+
+#################################################
+# set database
+rrna='/mnt/t64/test/sce/1.reference/rrna/rrna'
+trna='/mnt/t64/test/sce/1.reference/trna/trna'
+ncrna='/mnt/t64/test/sce/1.reference/ncrna/ncrna'
+mrna='/mnt/t64/test/sce/1.reference/mrna/mrna'
+chrom='/mnt/t64/test/sce/1.reference/genome/genome'
+
+# alignment reads to reference
+for fq in /mnt/t64/test/sce/4.ribo-seq/1.cleandata/*fastq.gz
+do
+fqname=`\basename $fq .fastq.gz`
+
+## rrna
+bowtie -p 10 -v 1 --un="$fqname".norrna.fq --al="$fqname".rrna.fq \
+ -x $rrna $fq -S "$fqname".rrna.sam 2>> "$fqname".log
+
+## trna
+bowtie -p 10 -v 1 --un="$fqname".notrna.fq --al="$fqname".trna.fq \
+ -x $trna "$fqname".norrna.fq -S "$fqname".trna.sam 2>> "$fqname".log
+
+## ncrna
+bowtie -p 10 -v 1 --un="$fqname".noncrna.fq --al="$fqname".ncrna.fq \
+ -x $ncrna "$fqname".notrna.fq -S "$fqname".ncrna.sam 2>> "$fqname".log
+
+## mrna
+bowtie -p 10 -v 1 --un="$fqname".nomrna.fq --al="$fqname".mrna.fq \
+ -x $mrna "$fqname".noncrna.fq -S "$fqname".mrna.sam 2>> "$fqname".log
+
+## genome
+bowtie -p 10 -v 1 --un="$fqname".nogenome.fq --al="$fqname".genome.fq 、
+ -x $chrom "$fqname".nomrna.fq -S "$fqname".genome.sam 2>> "$fqname".log
+
+## compress fastq
+pigz *fq
+
+## compress sam
+for sam in *.sam
+do
+samtools view -h -F 4 $sam | samtools sort -@ $threads -o `\basename $sam sam`bam
+rm $sam
+done
+
+done
+
+```
+
+4. 统计所有数据库的比对结果
+```bash
+#################################################
+# merge all log files
+merge_bwt_log \
+ -n rRNA,tRNA,ncRNA,mRNA,Genome \
+ -l *log -o sce
+
+```
+
+
+#### 3.1.4 使用 STAR 比对 mRNA 的 reads
+去除掉 ncRNA 的 reads 之后，使用 star 重新比对到酵母的基因组。
+
+1. 使用 star 比对 RNA-seq 的数据
+```bash
+cd /mnt/t64/test/sce/3.rna-seq/3.star/
+
+#################################################
+# set the option and database
+genome='/mnt/t64/test/sce/1.reference/star-index/'
+
+#################################################
+# map the all rna-seq reads to genome and transcriptome region
+for fastq in /mnt/t64/test/sce/3.rna-seq/2.bowtie/*.noncrna.fq.gz
+do
+
+## get file name
+output=$(basename $fastq .noncrna.fq.gz)
+
+#################################################
+## run the alignment
+STAR --runThreadN 10 \
+ --readFilesCommand zcat \
+ --genomeDir $genome \
+ --readFilesIn $fastq \
+ --outFileNamePrefix $output \
+ --outSAMtype BAM Unsorted \
+ --outFilterType BySJout \
+ --quantMode TranscriptomeSAM GeneCounts \
+ --outReadsUnmapped Fastx \
+ --outSAMattributes All \
+ --alignEndsType Local \
+ --outFilterMultimapNmax 3 \
+ --outFilterMismatchNmax 1 \
+ --alignIntronMax 10000 \
+ --outFilterMatchNmin 20
+# --outWigType wiggle --outWigNorm RPM
+
+pigz *mate1
+
+#################################################
+## sort the bam file
+samtools sort -@ 10 $output"Aligned.out.bam" -o $output"Aligned.sortedByCoord.out.bam"
+samtools index -@ 10 $output"Aligned.sortedByCoord.out.bam"
+rm $output"Aligned.out.bam"
+
+done
+```
+
+2. 使用 star 比对 Ribo-seq 的数据
+```bash
+cd /mnt/t64/test/sce/4.ribo-seq/3.star/
+
+#################################################
+# set the option and database
+genome='/mnt/t64/test/sce/1.reference/star-index/'
+
+#################################################
+# map the all rna-seq reads to genome and transcriptome region
+for fastq in /mnt/t64/test/sce/4.ribo-seq/2.bowtie/*.noncrna.fq.gz
+do
+
+## get file name
+output=$(basename $fastq .noncrna.fq.gz)
+
+#################################################
+## run the alignment
+STAR --runThreadN 10 \
+ --readFilesCommand zcat \
+ --genomeDir $genome \
+ --readFilesIn $fastq \
+ --outFileNamePrefix $output \
+ --outSAMtype BAM Unsorted \
+ --outFilterType BySJout \
+ --quantMode TranscriptomeSAM GeneCounts \
+ --outReadsUnmapped Fastx \
+ --outSAMattributes All \
+ --alignEndsType Local \
+ --outFilterMultimapNmax 3 \
+ --outFilterMismatchNmax 1 \
+ --alignIntronMax 10000 \
+ --outFilterMatchNmin 20
+# --outWigType wiggle --outWigNorm RPM
+
+pigz *mate1
+
+#################################################
+## sort the bam file
+samtools sort -@ 10 $output"Aligned.out.bam" -o $output"Aligned.sortedByCoord.out.bam"
+samtools index -@ 10 $output"Aligned.sortedByCoord.out.bam"
+rm $output"Aligned.out.bam"
+
+done
+```
+
+
+#### 3.1.5 使用 RSEM 或者 featureCounts 定量基因表达水平
+我们可以已使用 RSEM 或者 featureCounts来对基因的表达水平进行定量，二者各有特色，这里使用 RSEM 做示例。
+
+1. 定量 RNA-seq 的转录水平
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/4.quantification/
+
+#################################################
+# quantify the gene expression
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/*Aligned.toTranscriptome.out.bam
+do
+rsem-calculate-expression -p 10 --no-bam-output --alignments -q $bam /mnt/t64/test/sce/1.reference/rsem-index/sce `\basename $bam Aligned.toTranscriptome.out.bam`
+# rsem-calculate-expression -p 10 --paired-end --no-bam-output --alignments -q $bam /mnt/t64/test/sce/1.reference/rsem-index/sce `\basename $bam Aligned.toTranscriptome.out.bam`
+done
+```
+
+2. 合并 RNA-seq 的数据定量结果
+```bash
+#################################################
+# merge the gene expression
+merge_rsem -c expected_count -l *.genes.results -o gene.expected_count.txt
+merge_rsem -c TPM -l *.genes.results -o gene.TPM.txt
+merge_rsem -c FPKM -l *.genes.results -o gene.FPKM.txt
+
+#################################################
+# merge the isoforms expression
+merge_rsem -c expected_count -l *.isoforms.results -o isoforms.expected_count.txt
+merge_rsem -c TPM -l *.isoforms.results -o isoforms.TPM.txt
+merge_rsem -c FPKM -l *.isoforms.results -o isoforms.FPKM.txt
+
+```
+
+
+3. 定量 Ribo-seq 的转录水平
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/4.quantification/
+
+#################################################
+# quantify the isoforms expression
+for bam in /mnt/t64/test/sce/4.ribo-seq/3.star/*Aligned.toTranscriptome.out.bam
+do
+rsem-calculate-expression -p 10 --no-bam-output --alignments -q $bam /mnt/t64/test/sce/1.reference/rsem-index/sce `\basename $bam Aligned.toTranscriptome.out.bam`
+# rsem-calculate-expression -p 10 --paired-end --no-bam-output --alignments -q $bam /mnt/t64/test/sce/1.reference/rsem-index/sce `\basename $bam Aligned.toTranscriptome.out.bam`
+done
+```
+
+4. 合并 Ribo-seq 的数据定量结果
+```bash
+#################################################
+# merge the gene expression
+merge_rsem -c expected_count -l *.genes.results -o gene.expected_count.txt
+merge_rsem -c TPM -l *.genes.results -o gene.TPM.txt
+merge_rsem -c FPKM -l *.genes.results -o gene.FPKM.txt
+
+#################################################
+# merge the isoforms expression
+merge_rsem -c expected_count -l *.isoforms.results -o isoforms.expected_count.txt
+merge_rsem -c TPM -l *.isoforms.results -o isoforms.TPM.txt
+merge_rsem -c FPKM -l *.isoforms.results -o isoforms.FPKM.txt
+
+```
+
+
+### 3.2 使用 RiboParser 继续完成 GSE67387 的数据分析
+#### 3.2.1 测序数据的质量检查
+1. 检查 Ribo-seq 数据的测序质量
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/01.qc/
+
+#################################################
+# check the ribo-seq quality
+for bam in /mnt/t64/test/sce/4.ribo-seq/3.star/*Aligned.toTranscriptome.out.bam
+do
+prefix_name=$(basename $bam Aligned.toTranscriptome.out.bam)
+
+rpf_Check -b $bam -s --thread 10 -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+  -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+2. 合并所有样本的质量分析结果
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/
+
+#################################################
+# merge the ribo-seq quality results
+merge_length -l ./01.qc/*length_distribution.txt -o sce
+merge_saturation -l ./01.qc/*gene_saturation.txt -o sce
+
+```
+
+
+3. 检查 RNA-seq 数据的测序质量
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/01.qc/
+
+#################################################
+# check the ribo-seq quality
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/*Aligned.toTranscriptome.out.bam
+do
+prefix_name=$(basename $bam Aligned.toTranscriptome.out.bam)
+
+rpf_Check -b $bam -s --thread 10 -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+  -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+4. 合并所有样本的质量分析结果
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/
+
+#################################################
+# merge the rna-seq quality results
+merge_length -l ./01.qc/*length_distribution.txt -o sce
+merge_saturation -l ./01.qc/*gene_saturation.txt -o sce
+
+```
+
+
+#### 3.2.2 测序数据的酶切和酶连的偏好性
+1. 检查 Ribo-seq 数据的酶切和酶连的偏好性
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/02.digestion/
+
+#################################################
+# check the reads digestion
+for bam in /mnt/t64/test/sce/4.ribo-seq/3.star/01.qc/*.bam
+do
+prefix_name=$(basename $bam .bam)
+
+rpf_Digest -b $bam -m 27 -M 33 --scale \
+ -s /mnt/t64/test/sce/1.reference/norm/sce.norm.rna.fa \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+2. 合并所有样本的 reads digestion
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/
+
+#################################################
+# merge the rpf digestion
+merge_digestion -l ./02.digestion/*pwm.txt -o sce
+
+```
+
+
+3. 检查 RNA-seq 数据的酶切和酶连的偏好性
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/02.digestion/
+
+#################################################
+# check the reads digestion
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/01.qc/*.bam
+do
+prefix_name=$(basename $bam .bam)
+
+rpf_Digest -b $bam -m 25 -M 50 --scale \
+ -s /mnt/t64/test/sce/1.reference/norm/sce.norm.rna.fa \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+4. 合并所有样本的 reads digestion
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/
+
+#################################################
+# merge the rpf digestion
+merge_digestion -l ./02.digestion/*pwm.txt -o sce
+
+```
+
+
+#### 3.2.3 使用 RiboParser 做质量检查
+1. 预测 Ribo-seq 中的最佳 offset
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/03.offset/
+
+#################################################
+# predict the offset table
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/01.qc/*.bam
+do
+prefix_name=$(basename $bam .bam)
+
+rpf_Offset -b $bam -m 27 -M 33 -p 30 -d \
+ --mode RSBM \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+2. 合并所有样本的 offset 预测结果
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/
+
+#################################################
+# merge the ribo-seq offset results
+merge_offset_detail -l ./03.offset/*end.txt -o sce
+merge_offset -l ./03.offset/*sscbm_offset.txt -o sce_sscbm
+merge_offset -l ./03.offset/*rsbm_offset.txt -o sce_rsbm
+
+```
+
+3. RNA-seq 无需预测 offset，这里直接创建一个文件，其中 offset 值均为 12。
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/03.offset/
+
+#################################################
+# set the offset table
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/01.qc/*.bam
+do
+
+prefix_name=$(basename $bam .bam)
+rna_Offset -m 27 -M 50 -e 12 -o $prefix_name &> $prefix_name".log"
+
+done
+```
+
+
+#### 3.2.4 把 bam 文件中的 reads 转换为 txt 文件中的 density。
+1. 转换 Ribo-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/04.density/
+
+#################################################
+# convert the rpf to density
+for bam in /mnt/t64/test/sce/4.ribo-seq/3.star/01.qc/*.bam
+do
+prefix_name=$(basename $bam .bam)
+
+rpf_Density -b $bam -m 27 -M 33 --period 40 -l --thread 10 \
+ -p /mnt/t64/test/sce/4.ribo-seq/3.star/03.offset/$prefix_name"_rsbm_offset.txt" \
+ -s /mnt/t64/test/sce/1.reference/norm/sce.norm.rna.fa \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -o $prefix_name &> $prefix_name".log"
+
+done
+
+```
+
+2. 转换 RNA-seq 数据
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/04.density/
+
+#################################################
+# convert the reads to density
+for bam in /mnt/t64/test/sce/3.rna-seq/3.star/01.qc/*.bam
+do
+prefix_name=$(basename $bam .bam)
+
+rna_Density -b $bam -m 27 -M 33 --period 40 -l --thread 10 \
+ -p /mnt/t64/test/sce/3.rna-seq/3.star/03.offset/$prefix_name"_offset.txt" \
+ -s /mnt/t64/test/sce/1.reference/norm/sce.norm.rna.fa \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -o $prefix_name &> $prefix_name".log"
+
+done
+
+```
+
+
+#### 3.2.5 合并所有文件
+1. 合并 Ribo-seq density 文件
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/
+
+#################################################
+# create the samples file: Ribo.file.list
+merge_dst_list -l ../04.density/*_rpf.txt -o RPF.file.list
+
+
+cat RPF.file.list
+
+Name File  Type
+wt_ribo_YPD1	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944912_rpf.txt Ribo
+wt_ribo_YPD2	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944913_rpf.txt Ribo
+wt_ribo_YPD3	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944914_rpf.txt Ribo
+ncs2d_ribo_YPD1	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944915_rpf.txt Ribo
+ncs2d_ribo_YPD2	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944916_rpf.txt Ribo
+ncs2d_ribo_YPD3	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944917_rpf.txt Ribo
+elp6d_ribo_YPD1	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944918_rpf.txt Ribo
+elp6d_ribo_YPD2	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944919_rpf.txt Ribo
+elp6d_ribo_YPD3	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944920_rpf.txt Ribo
+ncs2d_elp6d_ribo_YPD1	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944921_rpf.txt Ribo
+ncs2d_elp6d_ribo_YPD2	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944922_rpf.txt Ribo
+ncs2d_elp6d_ribo_YPD3	/mnt/t64/test/sce/4.ribo-seq/04.density/SRR1944923_rpf.txt Ribo
+
+#################################################
+# merge all the Ribo-seq files
+rpf_Merge -l RPF.file.list -o sce_rpf &> sce.log
+
+```
+
+2. 合并 RNA-seq density 文件
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/
+
+#################################################
+# create the samples file: RNA.file.list
+merge_dst_list -l ../04.density/*_rna.txt -o RNA.file.list
+
+cat RNA.file.list
+
+Name File  Type
+wt_rna_YPD1	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944912_rna.txt RNA
+wt_rna_YPD2	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944913_rna.txt RNA
+wt_rna_YPD3	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944914_rna.txt RNA
+ncs2d_rna_YPD1	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944915_rna.txt RNA
+ncs2d_rna_YPD2	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944916_rna.txt RNA
+ncs2d_rna_YPD3	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944917_rna.txt RNA
+elp6d_rna_YPD1	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944918_rna.txt RNA
+elp6d_rna_YPD2	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944919_rna.txt RNA
+elp6d_rna_YPD3	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944920_rna.txt RNA
+ncs2d_elp6d_rna_YPD1	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944921_rna.txt RNA
+ncs2d_elp6d_rna_YPD2	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944922_rna.txt RNA
+ncs2d_elp6d_rna_YPD3	/mnt/t64/test/sce/3.rna-seq/04.density/SRR1944923_rna.txt RNA
+
+#################################################
+# merge all the RNA-seq files
+rpf_Merge -l RNA.file.list -o sce_rna &> sce.log
+
+```
+
+
+#### 3.2.6 计算三核苷酸周期性
+1. 检查 Ribo-seq 数据三核苷酸周期性
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/06.periodicity/
+
+#################################################
+# check the periodicity
+rpf_Periodicity \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 30 --tis 0 --tts 0 -o sce &> sce.log
+
+```
+
+2. 检查 RNA-seq 数据三核苷酸周期性
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/06.periodicity/
+
+#################################################
+# check the periodicity
+rpf_Periodicity \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -m 30 --tis 0 --tts 0 -o sce &> sce.log
+
+```
+
+
+#### 3.2.7 起始和终止密码子前后的 meta-gene 分析
+1. Ribo-seq 数据 meta-gene 分析
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/07.metaplot/
+
+#################################################
+# metagene analysis
+rpf_Metaplot \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 50 --mode bar -o sce &> sce.log
+
+```
+
+2. RNA-seq 数据 meta-gene 分析
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/07.metaplot/
+
+#################################################
+# metagene analysis
+rpf_Metaplot \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -m 50 --mode bar -o sce &> sce.log
+
+```
+
+
+#### 3.2.8 检查基因上的整体 density 覆盖情况
+1. 检查 Ribo-seq 数据的 density 覆盖
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/08.coverage/
+
+#################################################
+# check the rpf density along with the gene body
+rpf_Coverage \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 50 --outlier \
+ -b 10,100,10 \
+ -n --heat \
+ -o sce &> sce.log
+
+```
+
+2. 检查 RNA-seq 数据的 density 覆盖
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/08.coverage/
+
+#################################################
+# check the reads density along with the gene body
+rpf_Coverage \
+ -t /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -m 50 --outlier \
+ -b 10,100,10 \
+ -n --heat \
+ -o sce &> sce.log
+
+```
+
+
+#### 3.2.9 检查样本之间的重复性
+1. 检查 Ribo-seq 数据样本重复性
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/09.correlation/
+
+#################################################
+# calculate the samples replication of Ribo-seq
+rpf_Corr \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -o sce &> sce.log
+
+```
+
+2. 检查 RNA-seq 数据的重复性
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/09.correlation/
+
+#################################################
+# calculate the samples replication of RNA-seq
+rpf_Corr \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -o sce &> sce.log
+
+```
+
+
+#### 3.2.10 基因表达和翻译水平定量
+1. 计算基因的翻译量（RPFs level）
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/10.quantification/
+
+#################################################
+# quantify the gene expression
+rpf_Quant \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ --tis 15 \
+ --tts 5 \
+ -o sce &> sce.log 
+
+```
+
+
+#### 3.2.11 计算密码子水平的 pausing score
+1. 计算 Ribo-seq 数据中密码子水平的 pausing score
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/11.pausing_score/
+
+#################################################
+# calculate the codon pausing score of E/P/A site
+for sites in E P A
+do
+rpf_Pausing \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -b 0 --stop \
+ -m 30 \
+ -s $sites \
+ -f 0 \
+ --scale minmax \
+ -o "$sites"_site &> "$sites"_site.log
+done
+
+```
+
+
+#### 3.2.12 计算密码子水平的 occupancy
+1. 计算 Ribo-seq 数据中密码子水平的 occupancy
+```bash
+$ cd /mnt/t64/test/sce/4.rpf-seq/5.riboparser/12.codon_occupancy/
+
+#################################################
+# calculate the codon occupancy of E/P/A site
+for sites in E P A
+do
+rpf_Occupancy \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 30 \
+ -s "$sites" \
+ -f 0 --stop \
+ --scale minmax \
+ -o "$sites"_site &> "$sites"_site.log
+done
+
+```
+
+
+#### 3.2.13 计算密码子水平的 decoding time
+1. 计算 Ribo-seq 数据中密码子水平的 decoding time
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/13.codon_decoding_time/
+
+#################################################
+# calculate the codon decoding time of E/P/A site
+for sites in E P A
+do
+rpf_CDT \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ --rna /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ --rpf /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ --stop \
+ -m 50 \
+ -f 0 \
+ -s $sites \
+ --tis 10 \
+ --tts 5 \
+ -o "$sites"_site &> "$sites"_site.log
+done
+
+```
+
+
+#### 3.2.14 计算密码子水平的 selection time
+1. 计算 Ribo-seq 数据中密码子水平的 selection time
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/14.codon_selection_time/
+
+#################################################
+# calculate the codon selection time of E/P/A site
+for sites in E P A
+do
+rpf_CST \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ --rna /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ --rpf /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ --stop \
+ -m 50 \
+ -f 0 \
+ -s $sites \
+ --tis 10 \
+ --tts 5 \
+ -o "$sites"_site &> "$sites"_site.log
+done
+
+```
+
+
+#### 3.2.15 计算基因和密码子水平的变异系数
+1. 计算 Ribo-seq 数据中基因和密码子水平的变异系数
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/15.coefficient_of_variation/
+
+#################################################
+# Here we can configure the design file to calculate differences between different groups.
+$ cat design.txt
+name	group
+WT_ribo_YPD1	WT_ribo_YPD
+WT_ribo_YPD2	WT_ribo_YPD
+WT_ribo_YPD3	WT_ribo_YPD
+ncs2d_ribo_YPD1	ncs2d_ribo_YPD
+ncs2d_ribo_YPD2	ncs2d_ribo_YPD
+ncs2d_ribo_YPD3	ncs2d_ribo_YPD
+elp6d_ribo_YPD1	elp6d_ribo_YPD
+elp6d_ribo_YPD2	elp6d_ribo_YPD
+elp6d_ribo_YPD3	elp6d_ribo_YPD
+ncs2d_elp6d_ribo_YPD1	ncs2d_elp6d_ribo_YPD
+ncs2d_elp6d_ribo_YPD2	ncs2d_elp6d_ribo_YPD
+ncs2d_elp6d_ribo_YPD3	ncs2d_elp6d_ribo_YPD
+
+#################################################
+# calculate the coefficient of variation
+rpf_CoV \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -f 0 \
+ -m 30 \
+ --tis 10 \
+ --tts 5 \
+ --fig \
+ -g design.txt \
+ -o sce &> sce.log
+
+```
+
+#### 3.2.16 密码子 meta-codon 分析
+1. 计算 Ribo-seq 数据中密码子 meta density
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/16.meta_codon/
+
+#################################################
+# Here we can configure the codon list.
+$ cat codon_list.txt
+AAA
+AAC
+AAG
+AAT
+AAGAAG
+ATGATG
+CCCGGG
+...
+
+
+#################################################
+# codon meta analysis
+rpf_Meta_Codon \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 50 -f 0 \
+ -c codon_list.txt \
+ -a 15 -u -n --fig \
+ -o sce &> sce.log
+
+```
+
+#### 3.2.17 Data shuffling
+1. 重新洗牌 Ribo-seq 数据的 gene density 文件
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/17.shuffle/
+
+#################################################
+# codon meta analysis
+rpf_Shuffle \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -s 0 \
+ -i \
+ -o sce &> sce.log
+
+```
+
+2. 重新洗牌 RNA-seq 数据的 gene density 文件
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/11.shuffle/
+
+#################################################
+# retrieve and format the gene density
+rpf_Shuffle \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -s 0 \
+ -i \
+ -o sce &> sce.log
+
+```
+
+#### 3.2.18 提取 gene density
+1. 提取和格式化 Ribo-seq 数据中的 gene density
+```bash
+$ cd /mnt/t64/test/sce/4.ribo-seq/5.riboparser/18.gene_density/
+
+#################################################
+# codon meta analysis
+rpf_Retrieve \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/4.ribo-seq/5.riboparser/05.merge/sce_rpf_merged.txt \
+ -m 0 \
+ -f \
+ -n \
+ -o sce &> sce.log
+
+```
+
+2. 提取和格式化 RNA-seq 数据中的 gene density
+```bash
+$ cd /mnt/t64/test/sce/3.rna-seq/5.riboparser/12.gene_density/
+
+#################################################
+# retrieve and format the gene density
+rpf_Retrieve \
+ -l /mnt/t64/test/sce/1.reference/norm/sce.norm.txt \
+ -r /mnt/t64/test/sce/3.rna-seq/5.riboparser/05.merge/sce_rna_merged.txt \
+ -m 0 \
+ -f \
+ -n \
+ -o sce &> sce.log
+
+```
+
+
+## 4. 贡献
+
+欢迎提交问题和贡献代码
+联系 rensc0718@163.com
+
+## 5. 许可证
+
+本项目可免费用于学术研究，不得用于商业用途。