RNA_velocity

最新推荐文章于 2024-08-07 09:59:29 发布

lejinglife

最新推荐文章于 2024-08-07 09:59:29 发布

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本文链接：https://blog.csdn.net/lejinglife/article/details/129202060

版权

该文详细描述了如何处理和分析RNA测序数据，包括从NFS数据存储中下载和准备fastq文件，获取并解压缩参考基因组，构建Salmon和alevin-fry的索引，以及执行mapping和quantification步骤来获得RNA-velocity结果。

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数据存储

alevin-fry data:

/nfs_data/lidx/project/rna_velocity/fry_a/data/SRR9201794_1.fastq.gz

/nfs_data/lidx/project/rna_velocity/fry_a/data/SRR9201794_2.fastq.gz

An introduction to RNA-velocity using alevin-fry (combine-lab.github.io)

使用数据存储：

/nfs_data/lidx/project/rna_velocity/NB/data/SRR10156295_R1_fastq.gz

GEO Accession viewer (nih.gov)

# 代码

# download data
cd project/rna_velocity/NB/data

prefetch SRR10156295

fastq-dump --split-files --gzip SRR10156295

mv SRR10156295_2.fastq.gz SRR10156295_R1_fastq.gz

mv SRR10156295_3.fastq.gz SRR10156295_R2_fastq.gz

# get ref_genome
mkdir ref_data

cd /nfs_data/database/ref_genomes/human_GRCh38p13/ens107/

cp Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz /nfs_data/lidx/project/rna_velocity/NB/data/ref_data

cp Homo_sapiens.GRCh38.107.gtf.gz /nfs_data/lidx/project/rna_velocity/NB/data/ref_data

cd/nfs_data/lidx/project/rna_velocity/NB/data/ref_data

gunzip Homo_sapiens.GRCh38.107.gtf.gz
 
gunzip Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz 

##107报错，选了一个旧的版本
wget ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
gunzip Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz


wget ftp://ftp.ensembl.org/pub/release-93/gtf/homo_sapiens/Homo_sapiens.GRCh38.93.gtf.gz
gunzip Homo_sapiens.GRCh38.93.gtf.gz





# making splici refernce using 
cd ..

pyroe make-splici \
ref_data/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
ref_data/Homo_sapiens.GRCh38.107.gtf \
151 GRCh38_splici_fl146 \
--flank-trim-length 5 --filename-prefix splici
####107报错，换了93版本

# make gene ID to gene name mapping file
gffread ref_data/Homo_sapiens.GRCh38.93.gtf -o ref_data/Homo_sapiens.GRCh38.93.gff

grep "gene_name" ref_data/Homo_sapiens.GRCh38.93.gff | cut -f9 | cut -d';' -f2,3 | sed 's/=/ /g' | sed 's/;/ /g' | cut -d' ' -f2,4 | sort | uniq > GRCh38_geneid_to_name.txt


# Building the salmon/alevin splici index
cd ..

salmon index -t data/GRCh38_splici_fl146/splici_fl146.fa -i GRCh38_splici_fl146_idx -p 16


# Mapping the data to obtain a RAD file
$ salmon alevin -i GRCh38_splici_fl146_idx -p 16 -l ISR --chromium --sketch \
-1 data/SRR10156295_R1.fastq.gz \
-2 data/SRR10156295_R2.fastq.gz \
-o NB_map 

# Processing the mapped reads
$ alevin-fry generate-permit-list -d fw -k -i NB_map -o NB_quant
$ alevin-fry collate -t 16 -i NB_quant -r NB_map
$ alevin-fry quant -t 16 -i NB_quant -o NB_quant_res --tg-map data/GRCh38_splici_fl146/splici_fl146_t2g_3col.tsv --resolution cr-like --use-mtx

$ alevin-fry generate-permit-list -d fw -k -i NB_map -o NB_quant1
$ alevin-fry collate -t 16 -i NB_quant1 -r NB_map
$ alevin-fry quant -t 16 -i NB_quant1 -o NB_quant1_res --tg-map data/GRCh38_splici_fl146/splici_fl146_t2g_3col.tsv --resolution cr-like --use-mtx