rnaseq分析中片段inner size出现问题

片段inner size，片段选择是否异常

输入命令后：for i in DL125NS1 DL125NS2 DL125NS3 DL125PS1 DL125PS2 DL125PS3; do

echo "RUN CMD: inner_distance.py -i $workdir/3.map/hisat2/${i}.bam -r $GENE_BED -o ${i}_inner_size"

inner_distance.py -i $workdir/3.map/hisat2/${i}.bam -r $GENE_BED -o ${i}_inner_size

done

命令输出：

> echo "RUN CMD: inner_distance.py -i $workdir/3.map/hisat2/${i}.bam -r $GENE_BED -o ${i}_inner_size"

> inner_distance.py -i $workdir/3.map/hisat2/${i}.bam -r $GENE_BED -o ${i}_inner_size

> done

RUN CMD: inner_distance.py -i /work/zhaoran_rnaseq/125PS_125NS/3.map/hisat2/DL125NS1.bam -r /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed -o DL125NS1_inner_size

Get exon regions from /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed ...

Load BAM file ... Done

Total read pairs used 0

Cannot find paired reads

RUN CMD: inner_distance.py -i /work/zhaoran_rnaseq/125PS_125NS/3.map/hisat2/DL125NS2.bam -r /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed -o DL125NS2_inner_size

Get exon regions from /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed ...

Load BAM file ... Done

Total read pairs used 0

Cannot find paired reads

RUN CMD: inner_distance.py -i /work/zhaoran_rnaseq/125PS_125NS/3.map/hisat2/DL125NS3.bam -r /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed -o DL125NS3_inner_size

Get exon regions from /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed ...

Load BAM file ... Done

Total read pairs used 0

Cannot find paired reads
...

跑单个样本：

输入命令：inner_distance.py -i $workdir/3.map/hisat2/DL125NS1.bam -r $GENE_BED -o DL125NS1_inner_size

命令输出：Get exon regions from /work/zhaoran_rnaseq/125PS_125NS/ref/gene.bed ...

Load BAM file ... Done

Total read pairs used 0

Cannot find paired reads

尝试使用openai解决问题，samtools view 命令检查BAM文件是否包含配对的读段

samtools view $workdir/3.map/hisat2/DL125NS1.bam | grep -v '^@' | awk '{if ($2 == 77) print $0}' | wc -l

42637912
求老师帮助！

0 条评论
分类：转录组

默认排序时间排序

2 个回答

omicsgene - 生物信息 2024-05-21 09:52

擅长：重测序,遗传进化,转录组,GWAS

没有找到成对read的比对，你这个转录组是不是单端测序，单端测序没法评估这个；

或者你hisat比对的时候，输入read1文件和read2 文件，错误的都是输入read1 文件或者都是输入的read2 文件，检查命令行是否错误；

gongqinglong 2024-05-21 15:34

老师您好，我的数据是双端测序。代码经过比对也没发现问题。下面是我根据课程pipline改的操作流程代码和sam文件、bam文件里的部分内容，麻烦老师评估一下是哪里出了问题，非常感谢您：

#hisat2软件，链特异性文库设置

#--rna-strandness RF or FR

#非链特异性文库，不设置此参数

for i in PBS1 PBS2 PBS3 DL125PS1 DL125PS2 DL125PS3; do

echo "RUN CMD: hisat2 -p 1 --rg-id=${i} --rg SM:${i} --rg LB:${i} --rg PL:ILLUMINA \

-x $REF_INDEX --dta \

-1 $workdir/2.data_qc/${i}_1.clean.fq.gz \

-2 $workdir/2.data_qc/${i}_2.clean.fq.gz \

-S ${i}.sam 2>${i}.summary"

hisat2 -p 1 --rg-id=${i} --rg SM:${i} --rg LB:${i} --rg PL:ILLUMINA \

-x $REF_INDEX --dta --rna-strandness RF \

-1 $workdir/2.data_qc/${i}_1.clean.fq.gz \

-2 $workdir/2.data_qc/${i}_2.clean.fq.gz \

-S ${i}.sam 2>${i}.summary

done

# sam 转换成bam 格式并排序

for i in PBS1 PBS2 PBS3 DL125PS1 DL125PS2 DL125PS3; do

echo "RUN CMD: samtools sort --threads 8 -m 30G -o ${i}.bam ${i}.sam"

samtools sort --threads 8 -m 30G -o ${i}.bam ${i}.sam

done

# bam index建立索引

for i in PBS1 PBS2 PBS3 DL125PS1 DL125PS2 DL125PS3; do

echo "RUN CMD: samtools index ${i}.bam"

samtools index ${i}.bam

done

输出结果：

total 200G

-rw-r--r-- 1 root root 5.1G May 20 02:03 DL125_1.bam

-rw-r--r-- 1 root root 250K May 20 03:22 DL125_1.bam.bai

-rw-r--r-- 1 root root 29G May 19 22:10 DL125_1.sam

-rw-r--r-- 1 root root 610 May 19 22:10 DL125_1.summary

-rw-r--r-- 1 root root 4.8G May 20 02:05 DL125_2.bam

-rw-r--r-- 1 root root 250K May 20 03:23 DL125_2.bam.bai

-rw-r--r-- 1 root root 28G May 19 22:23 DL125_2.sam

-rw-r--r-- 1 root root 610 May 19 22:23 DL125_2.summary

-rw-r--r-- 1 root root 5.2G May 20 02:08 DL125_3.bam

-rw-r--r-- 1 root root 250K May 20 03:25 DL125_3.bam.bai

-rw-r--r-- 1 root root 30G May 19 22:37 DL125_3.sam

-rw-r--r-- 1 root root 610 May 19 22:37 DL125_3.summary

-rw-r--r-- 1 root root 4.6G May 20 02:11 DL125PS1.bam

-rw-r--r-- 1 root root 250K May 20 03:26 DL125PS1.bam.bai

-rw-r--r-- 1 root root 28G May 19 22:50 DL125PS1.sam

-rw-r--r-- 1 root root 610 May 19 22:50 DL125PS1.summary

-rw-r--r-- 1 root root 5.1G May 20 02:14 DL125PS2.bam

-rw-r--r-- 1 root root 250K May 20 03:27 DL125PS2.bam.bai

-rw-r--r-- 1 root root 31G May 19 23:05 DL125PS2.sam

-rw-r--r-- 1 root root 610 May 19 23:05 DL125PS2.summary

-rw-r--r-- 1 root root 4.7G May 20 02:16 DL125PS3.bam

-rw-r--r-- 1 root root 250K May 20 03:28 DL125PS3.bam.bai

-rw-r--r-- 1 root root 28G May 19 23:18 DL125PS3.sam

-rw-r--r-- 1 root root 610 May 19 23:18 DL125PS3.summary

rnaseq分析中片段inner size出现问题

2 个回答

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