pipeline——（2）

pipeline——（2）

step 1 生成index

star.sh

STAR \
--runMode genomeGenerate \
--runThreadN  20 \
--genomeFastaFiles /share/pub/wangxy/software/genome/ucsc/hg38/hg38.fa \
--genomeDir /share2/pub/yangjy/yangjy/database/STAR_index99 \
--sjdbGTFfile /share/pub/wangxy/Annotation/hg38/gencode.v34.annotation.gtf \
--sjdbOverhang 99

step 2 生成bam

bamGSE99248.sh

num_1="_1"
num_2="_2"
for file in [.FASTQ]
do
echo $file
STAR \
--runThreadN  6 \
--genomeDir /share2/pub/yangjy/yangjy/database/STAR_index99 \
--readFilesIn /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/fastq_data/$file$num_1.fastq /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/fastq_data/$file$num_2.fastq \
--outFileNamePrefix /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/bam/$file \
--outFilterMultimapNmax 500 \
--outSAMtype BAM SortedByCoordinate \
--outSAMattributes NH HI NM MD XS AS
done

step 3 组装转录本

assembl.sh

tailfix="Aligned.sortedByCoord.out"
for file in [fastq names list]
do
echo $file$tailfix
stringtie /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/bbam/$file$tailfix.bam -j 2 -s 5 -f 0.05 -c 2 -p 5 \
-A /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/$file.gene_abund.tab \
-o /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/$file.out.gtf  
done

step 4 merge组装的转录本

ls -R /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/*.out.gtf > tmp.txt

taco_run.sh

/share2/pub/yangjy/yangjy/softs/taco-v0.7.3.Linux_x86_64/taco_run -o /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/merged_stringtie -p 10 /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/gtf_to_merge.txt

step 5 生成saf文件

export_saf.sh

cd /share2/pub/yangjy/yangjy/softs/TE_expression_in_scRNAseq/scripts/
/share2/pub/yangjy/yangjy/conda3/envs/qapa/bin/Rscript /share2/pub/yangjy/yangjy/softs/TE_expression_in_scRNAseq/scripts/export_saf_files.r -g /share2/pub/yangjy/yangjy/database/hg38_refGene.txt -r /share2/pub/yangjy/yangjy/database/hg38_rmsk.txt -l /share2/pub/yangjy/yangjy/database/STAR_index99/chrNameLength.txt -n /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/protemp/hg38 -m chrM -p 10

step 6 选择TE转录本

process_ass_trans.sh

cd /share2/pub/yangjy/yangjy/softs/TE_expression_in_scRNAseq/scripts/
/share2/pub/yangjy/yangjy/conda3/envs/qapa/bin/Rscript /share2/pub/yangjy/yangjy/softs/TE_expression_in_scRNAseq/scripts/processing_assembled_transcripts.r -f /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/merged_stringtie/assembly.gtf -g /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/protemp/hg38_pc_exon.saf -a /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/protemp/hg38_nc_exon.saf -m /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/protemp/hg38_chrM.saf -r /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/protemp/hg38_te.saf -n /share2/pub/yangjy/yangjy/rna-seq-data/GSE99248/assembl/result/hg38 -p 10

KO！