耗时：⼀个25G的sam⽂件整个流程⼤概15mins，我这⾥6个sam⼤概160G，总耗时约2hrBAM⽂件质控（可选）2022-2-16软件RSeQC RNA-Seq质控⼯具，多个python包，⽤于评估RNA-seq的数据：检查序列质量、核酸组分偏性、PCR偏性、GC含量偏性，还有RNA-seq特异性模块：评估测序饱和度、映射读数分布、覆盖均匀性、链特异性、转录⽔平RNA完整性等 参数#⽐对情况Usage: bam_ [options]Summarizing mapping statistics of a BAM or SAM s: --version show program's version number and exit -h, --help show this help message and exit -i INPUT_FILE, --input-file=INPUT_FILE Alignment file in BAM or SAM format. -q MAP_QUAL, --mapq=MAP_QUAL Minimum mapping quality (phred scaled) to determine "uniquely mapped" reads. default=30#基因组覆盖率Usage: read_ [options]Check reads distribution over exon, intron, UTR, intergenic ... etcThe following reads will be skipped: qc_failed PCR duplicate Unmapped Non-primary (or secondary)Options: --version show program's version number and exit -h, --help show this help message and exit -i INPUT_FILE, --input-file=INPUT_FILE Alignment file in BAM or SAM format. -r REF_GENE_MODEL, --refgene=REF_GENE_MODEL Reference gene model in bed format.#需要bed⽂件#下载bed⽂件/projects/rseqc/files/BED/Human_Homo_sapiens/hg38_/download#本地下载解压缩，将bed⽂件上传服务器#这个下载链接有时候没反应，科学上⽹后就能正常 ⽤法for i in {61,62,63,191,193,194}dobam_ -i /mnt/AGS_RNA-seq/bam_sort/${i}. > /mnt/AGS_RNA-seq/scripts_log/${i}_eread_ -i /mnt/AGS_RNA-seq/bam_sort/${i}. -r /mnt/AGS_RNA-seq/BED_file/hg38_ > /mnt/AGS_RNA-seq/scripts_log/${i}_distribCount：FeaturecountsRNA-seq的count有许多可选的软件，HTseq、Stringtie、featurecounts，看教程⾥HTseq和featurecounts的结果差异不⼤，但HTseq耗时太久，所以选择featurecounts进⾏下⼀步安装conda install subread⽂件准备输⼊⽂件需要⼀个注释⽂件和bam_sorted#下载注释⽂件cd /mnt/AGS_RNA-seq/GTFaxel /pub/current_gtf/homo_sapiens/Homo_ -d Homo_使⽤for i in {61,62,63,191,193,194}dodone# -T 使⽤的线程数# -p 如果是paird end 就⽤# -t 将exon作为⼀个feature# -g 将gene_id作为⼀个feature# -a 参考的gtf/gff# -o 输出⽂件# 最后加上bam⽂件，注意bam⽂件要为sorted#全部合在⼀个counts⽂件featureCounts -T 16 -p -t exon -g gene_id -a /mnt/AGS_RNA-seq/GTF/Homo_ -o /mnt/AGS_RNA-seq/featureCounts/${i}. /mnt/featureCounts -T 16 -p -t exon -g gene_id -a /mnt/AGS_RNA-seq/GTF/Homo_ -o /mnt/AGS_RNA-seq/featureCounts/ /mnt/A其他参数 输出结果 ========== _____ _ _ ____ _____ ______ _____

===== / ____| | | | _ | __ | ____| / | __

===== | (___ | | | | |_) | |__) | |__ / | | | | ==== ___ | | | | _ <| _ /| __| / / | | | | ==== ____) | |__| | |_) | | | |____ / ____ | |__| | ========== |_____/ ____/|____/|_| _______/_/ ______/ v2.0.1//========================== featureCounts setting ===========================|| |||| Input files : 1 BAM file |||| o |||| |||| Output file : |||| Summary : y |||| Annotation : Homo_ (GTF) |||| Dir for temp files : /mnt/AGS_RNA-seq/featureCounts |||| |||| Threads : 16 |||| Level : meta-feature level |||| Paired-end : yes |||| Multimapping reads : not counted |||| Multi-overlapping reads : not counted |||| Min overlapping bases : 1 |||| |||| Chimeric reads : counted |||| Both ends mapped : not required |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 30720085 |||| Successfully assigned alignments : 22645654 (73.7%) |||| Running time : 0.52 minutes |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 27504748 |||| Successfully assigned alignments : 19565202 (71.1%) |||| Running time : 0.40 minutes |||| |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 27341548 |||| Successfully assigned alignments : 17090457 (62.5%) |||| Running time : 0.37 minutes |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 34769594 |||| Successfully assigned alignments : 21749916 (62.6%) |||| Running time : 0.89 minutes |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 38580695 |||| Successfully assigned alignments : 22749538 (59.0%) |||| Running time : 0.78 minutes |||| Running time : 0.78 minutes |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================////================================= Running ==================================|| |||| Load annotation file Homo_ ... |||| Features : 1552026 |||| Meta-features : 61541 |||| Chromosomes/contigs : 47 |||| |||| Process BAM file ... |||| Paired-end reads are included. |||| Total alignments : 37682170 |||| Successfully assigned alignments : 27515658 (73.0%) |||| Running time : 0.56 minutes |||| |||| Write the final count table. |||| Write the read assignment summary. |||| |||| Summary of counting results can be found in file "/mnt/AGS_RNA-seq/featur |||| eCounts/y" |||| ||============================================================================//Stringtie#stringtie为转录本拼接和定量的软件#利⽤stringtie分别输出gene count数⽬和FPKM值，gene count数⽤户基因差异分析，FPKM值为基因表达值，⽤于heatmap等绘制#Usage: stringtie [options]#aligned_ 是输⼊⽂件，该输⼊⽂件要求必须按基因组位置排序#参数-o []<>，设置StringTie组装转录本的输出GTF⽂件的路径和⽂件名。#参数-p 指定组装转录本的线程数（CPU）#参数-G 使⽤参考注释基因⽂件指导组装过程，格式GTF/GFF3。输出⽂件中既包含已知表达的转录本，也包含新的转录本。#参数-B 应⽤该选项，则会输出Ballgown输⼊表⽂件（* .ctab），其中包含⽤-G选项给出的参考转录本的覆盖率数据。#参数-A 输出基因丰度的⽂件（制表符分隔格式）#简写代码：stringtie {$i}. -o path/ -p 16 -G -eB -A path/#此次主要输出两个⽂件：gtf⽂件⽤于转换gene count⽂件；tab⽂件包含FPKM数值。————————————————版权声明：本⽂为CSDN博主「垚垚爸爱学习」的原创⽂章，遵循CC 4.0 BY-SA版权协议，转载请附上原⽂出处链接及本声明。原⽂链接：/xiaomotong123/article/details/106856233#参数-e 限制reads⽐对的处理，仅估计和输出与⽤-G选项给出的参考转录本匹配的组装转录本。使⽤该选项，则会跳过处理与参考转录本不匹配的组装转录本，这将⼤for i in {61,62,63,191,193,194}domkdir /mnt/AGS_RNA-seq/Stringtie/${i}donestringtie /mnt/AGS_RNA-seq/bam_sort/${i}. -o /mnt/AGS_RNA-seq/Stringtie/${i}/${i}.gtf -p 16 -G /mnt/AGS_RNA-seq/GTF/Homo_38.105.g