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一、SNP_spilt（鉴定亲本的等位基因在子代中特异性表达情况）

1.简介

2016年，Babraham生物信息组织发布了一款专门用来区分亲本来源reads的软件SNPsplit,它通过SAM/BAM文件reads上覆盖的已知SNP位点信息，
能够将reads分配给其中一个等位基因。
SNPsplit软件只需要提供用来区分印记来源的SNP信息，就可以针对生信常用软件（包括Bowtie2, TopHat, STAR, HISAT2, HiCUP和Bismark）比对后的bam区分其亲本来源。
参考链接：https://felixkrueger.github.io/SNPsplit/          
https://github.com/FelixKrueger/SNPsplit
https://f1000research.com/articles/5-1479/v2?s3BucketUrl=https%3A%2F%2Ff1000research.s3.amazonaws.com&gtmKey=GTM-PCBS9JK&submissionUrl=%2Ffor-authors%2Fpublish-your-research&otid=1bc074d1-3db4-47ed-9f80-df1a4a3f2ab4&immUserUrl=https%3A%2F%2Ff1r-proxy.f1krdev.com%2Feditor%2Fmember%2Fshow%2F

2.其分析过程如下

2.1 vcf的合并以及参考基因组的构建

#生成索引等操作

''samtools_path="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/QC/bin/samtools"
picard_jar="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/QC/share/picard-2.23.3-0/picard.jar"
"$samtools_path" faidx "$reference_genome_dir/reference_genome.fa"
java -jar "$picard_jar" CreateSequenceDictionary \
R="$reference_genome_dir/reference_genome.fa" \
O="$reference_genome_dir/reference_genome.dict"''

#合并vcf

''gatk_path="/PUBLIC/software/RNA/GATK/GenomeAnalysisTK.jar"
java -XX:ParallelGCThreads=4 -Xmx20g -jar "$gatk_path" \
-T CombineVariants \
-R "$reference_genome_dir/reference_genome.fa" \     
-V "$parent_vcf_dir/${parent1}_SNP.vcf" \
-V "$parent_vcf_dir/${parent2}_SNP.vcf" \
-o "$result_dir/${parent1}_${parent2}_all.vcf"''

#生成N掩盖基因组
''cd "$result_dir"
snp_split_path="/TJPROJ6/RNA_SH/personal_dir/fengjie/SOFTWARE/CONDA/conda/envs/SNPsplit/bin/SNPsplit_genome_preparation"
$snp_split_path \
--vcf_file "$result_dir/${parent1}_${parent2}_all.vcf" \
--strain "$parent1" \
--dual_hybrid \
--strain2 "$parent2" \
--reference_genome "$reference_genome_dir" \
--genome_build Human  # 这里需要根据实际情况更改''
#整理文件
''dual_hybrid_dir="${parent1}_${parent2}_dual_hybrid.based_on_Human_N-masked"
cd "$result_dir/$dual_hybrid_dir"
cat *.fa > all_N-masked.Human.N-masked.fa''

# 构建索引新基因组
''hisat2_build_path="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/QC/bin/hisat2-build"
"$hisat2_build_path" \
"$result_dir/$dual_hybrid_dir/all_N-masked.Human.N-masked.fa" \
"$result_dir/$dual_hybrid_dir/all_N-masked.Human.N-masked"''

2.2 比对排序

#比对

''hisat2_path="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/QC/bin/hisat2"
samtools_path="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/QC/bin/samtools"

cd "$bam_dir"

"$hisat2_path" -x "$result_dir/$dual_hybrid_dir/all_N-masked.Human.N-masked" \
-p 8 --dta -t --phred33 \
-1 "$fq_dir/$(basename "$fq1")" \
-2 "$fq_dir/$(basename "$fq2")" \
--no-softclip \
--un-conc-gz "${fq_name}.unmap.fq.gz" \
2> "${fq_name}_align.log" | \
"$samtools_path" sort -O BAM --threads 8 -o "${fq_name}.bam" -''

#拆分BAM文件
''snp_split_tool="/TJPROJ6/RNA_SH/personal_dir/fengjie/SOFTWARE/CONDA/conda/envs/SNPsplit/bin/SNPsplit"
"$snp_split_tool" \
--snp_file "$result_dir/${parent1}_${parent2}_all_SNPs_${parent1}_reference.based_on_Human.txt" \
-o "$sort_bam_dir" \
--paired --no_sort --singletons "${fq_name}.bam"''

2.4 转录组的定量以及画图

''export PYTHONPATH=""
unset PERL5LIB
export PATH="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/Quant/bin:$PATH"
export PATH="/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/r_3.5.0/bin:$PATH"
ln -s "$gtf_file" "$reference_genome_dir"
ln -s "$gene_file" "$reference_genome_dir"
cd "$quant_dir"''

# 生成feature_count

''featureCounts_path="/path/to/featureCounts"  # 请替换为实际路径，例如 /usr/bin/featureCounts
featureCounts -T 8 -F GTF -t exon -g gene_id -s 0 -Q 20 -C -B -p -a \
    "$reference_genome_dir/genome.gtf" \
    -o "$quant_dir/feature_count.xls" \
    "$sort_bam_dir/${fq_name}.genome1.bam" \
    "$sort_bam_dir/${fq_name}.genome2.bam"''
# 生成样本信息表
''get_condition_info="/TJPROJ7/GB_TR/PUBLIC/source/ncRNA/gb_tr_man_pipline/bin/get_condition_info"
"$get_condition_info" \
    --s2g "${fq_name}.genome1,${fq_name}.genome2" \
    --group "$parent1,$parent2" \
    --outfile "$quant_dir/condition.xls"''

# 生成count

''get_gene_count="/TJPROJ7/GB_TR/PUBLIC/source/ncRNA/gb_tr_man_pipline/bin/get_gene_count"
"$get_gene_count" \
--sample "${fq_name}.genome1,${fq_name}.genome2" \
--quant featureCounts \
--count "$quant_dir/feature_count.xls" \
--gene "$reference_genome_dir/genome_gene.xls" \
--outfile "$quant_dir/gene_count.xls"''
# 生成FPKM
''get_gene_fpkm="/TJPROJ7/GB_TR/PUBLIC/source/ncRNA/gb_tr_man_pipline/bin/get_gene_fpkm"
"$get_gene_fpkm" \
--count "$quant_dir/gene_count.xls" \
--condition "$quant_dir/condition.xls" \
--outfile "$quant_dir/gene_fpkm.xls"''

# 绘图

''Rscript_path="Rscript"  # 请根据实际情况修改
Rscript_script="/TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/shi_1.R"  # 确保脚本路径正确

"$Rscript_path" "$Rscript_script" \
    --inputfile "$quant_dir/gene_count.xls" \
    --outfile "$quant_dir/retu.pdf"''

参考脚本路径：/TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/SNP_spilt_1.sh

脚本帮助信息：

sh SNP_spilt_1.sh --help
用法: SNP_spilt_1.sh     --genome_fa <基因组文件路径>     --gtf <gtf文件路径>    
 --gene_file <gene文件路径>     --vcf1 <父本的vcf文件路径>     --vcf2 <母本的vcf文件路径>    
 --parent1 <父本名称>     --parent2 <母本名称> --fq1 <fq文件1路径> --fq2 <fq文件2路径>   
  --fq_name <fq文件名称>     --output_dir <输出文件夹路径>

脚本使用实例：

''sh SNP_spilt_1.sh --genome_fa /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/genome.fa\
    --gtf /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/reference_genome/genome.gtf\
    --gene_file /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/reference_genome/genome_gene.xls\
    --vcf1 /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/pepline_vcf/F1_SNP.vcf\
    --vcf2 /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/pepline_vcf/F2_SNP.vcf \
    --parent1 F1 \
    --parent2 F2 \
    --fq1 /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/fq.gz/T1/T1_1.fq.gz \
    --fq2 /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/data/fq.gz/T1/T1_2.fq.gz \
    --fq_name T1 \
    --output_dir /TJPROJ6/RNA_SH/personal_dir/zhengliuchang/yanfa2/ceshi
''

输出结果：
1.根据双亲snp比对后生成的bam文件：

T1.genome2.bam  T1.genome2.bam

2.gene_count文件
gene_count.xls

3.特异性热图
retu.pdf retu.png

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