smart-seq建库特点，从rna反转录成cDNA后，需要进行pcr扩增才能达到建库所需要核酸量，因此插入片段中有引物序列，需要使用cutadapt软件去除引物序列，命令如下：

cutadapt -g AAGCAGTGGTATCAACGCAGAGTAC -G AAGCAGTGGTATCAACGCAGAGTAC --error-rate 0.1 --overlap 10 -m 50 -o /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_1/vavcre2_1_1.primer.clean.fq.gz -p /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_1/vavcre2_1_2.primer.clean.fq.gz /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_1/vavcre2_1_1.clean.fq.gz /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_1/vavcre2_1_2.clean.fq.gz > /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_1/vavcre2_1_primer.log

然后按照qc流程分析（项目流程有更改，1.6中是raw先cutadapt然后在过fastp，目前流程/TJPROJ2/GB/PUBLIC/source/GB_TR/mRNA/gb_trans/gb_MedRef_man_pipline/auto_refpipline中，在是先得到clean再cutadapt，gc图需要看在哪里绘制，如果为去除引物前就绘制了，则可能发生GC抖动，一般是前30多bp，这是Novo试剂盒导致的，使用诺唯赞试剂盒则可以避免），两个流程fastp参数也有改变。

1.6如下：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/fastp-master/centos/fastp -G -q 20 -u 50 -n 0 -l 150 -j /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/1.6/QC/vavcre2_2/vavcre2_2.json -i /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/1.6/QC/vavcre2_2/vavcre2_2_1.fq.gz -I /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/1.6/QC/vavcre2_2/vavcre2_2_2.fq.gz -o /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/1.6/QC/vavcre2_2/vavcre2_2_1.clean.fq.gz -O /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/1.6/QC/vavcre2_2/vavcre2_2_2.clean.fq.gz

新版如下：

fastp --qualified_quality_phred 5 --unqualified_percent_limit 50  --n_base_limit 15 --min_trim_length 10 --overlap_len_require 30 --overlap_diff_limit 1 --overlap_diff_percent_limit 10  --length_required 150 --length_limit 150 --trim_poly_g --thread 1 --html /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_3/vavcre2_3_qc_report.html -j /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_3/vavcre2_3.json -i /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_3/vavcre2_3_1.fq.gz -I /TJPROJ6/RNA_SH/personal_dir/zhangxin/test/0518/Archive/BackUP/PJ/TJPROJ6/GB_TR/PJ_GB/mRNA/med/2001/andong/X101SC19120979-Z01-J231.B231-16_Mus.20220413/backup/Back_up/new/QC/vavcre2_3/vavcre2_3_2.fq.gz

相对来说，修改后的参数较1.6参数，阈值更加宽松，老师使用fastq软件在评估的过程中，可能出现红叉不合格的情况，则可使用1.6中的参数跑clean，再对clean进行评估。先进行cut后，reads的长度不足150，需要注意–length_required 150 –length_limit 150这两个参数，会过滤掉cut后的reads（这样则会有可能用novo试剂盒GC也是平滑的）

使用Novo试剂盒，GC图如下：

使用Novo试剂盒，cut 引物后，GC图如下：

使用Novo试剂盒，cut 引物后仅报错长度为150bp的reads（即丢掉含有引物的reads），GC图如下：

使用诺唯赞试剂盒，直接绘制GC图（没有对引物进行任何操作），如下图：