原始數(shù)據(jù)查看：
grep '^@' ctr.R1.fq | sort |uniq|wc -l
27787625 也就是乘以四后的行數(shù)遇八，即fq的name不會重
比對：
index=/media/pc/disk1/sun/refdata/ensembl_GRCm38/03_bowtie2_index/GRCm38
bowtie2 -p 20 -x $index -1 ctr.R1.fq -2 ctr.R2.fq -S ctr.sam &&
bowtie2 -p 20 -x $index -1 ko.R1.fq -2 ko.R2.fq -S ko.sam
比對結(jié)果：
查看sam行數(shù)：
igv:
samtools sort --threads 10 -m 2G -o?ctr.bam?ctr.sam &&
samtools sort --threads 10 -m 2G -o??ko.bam? ko.sam
samtools index?ctr.bam &&
samtools index?ko.bam
提取mapped：
samtools view -h -@ 10 -F 4 ctr.sam > ctr.mapped.sam &&
samtools view -h -@ 10 -F 4 ko.sam > ko.mapped.sam
wc -l 查看mapped的sam行數(shù)：
對mapped的進行igv:
samtools sort --threads 10 -m 3G -o?ctr.mapped.bam?ctr.mapped.sam &&
samtools sort --threads 10 -m 3G -o??ko.mapped.bam? ko.mapped.sam &&
samtools index?ctr.mappedbam &&
samtools index?ko.mapped.bam
#################################
trim:
trimmomatic PE -phred33 -threads 20 ctr.R1.fq ctr.R2.fq ctr.R1.paired.fq ctr.R1.unpaired.fq ctr.R2.paired.fq ctr.R2.unpaired.fq ILLUMINACLIP:/home/pc/miniconda3/pkgs/trimmomatic-0.38-1/share/trimmomatic-0.38-1/adapters/TruSeq3-PE.fa:2:30:10:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 AVGQUAL:20 MINLEN:36
trimmomatic PE -phred33 -threads 20?ko.R1.fq?ko.R2.fq?ko.R1.paired.fq?ko.R1.unpaired.fq?ko.R2.paired.fq ko.R2.unpaired.fq ILLUMINACLIP:/home/pc/miniconda3/pkgs/trimmomatic-0.38-1/share/trimmomatic-0.38-1/adapters/TruSeq3-PE.fa:2:30:10:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 AVGQUAL:20 MINLEN:36?
比對：
bowtie2 -p 20 -x $index -1 ctr.R1.paired.fq -2 ctr.R2.paired.fq -S ctr.paired.sam &&
bowtie2 -p 20 -x $index -1 ko.R1.paired.fq -2 ko.R2.paired.fq -S ko.paired.sam
經(jīng)過trim后比對情況并未改善：
1.fatsquniq：
網(wǎng)站：https://sourceforge.net/projects/fastuniq/
用法：
去重命令：
fastuniq -i list -o fastuniq.ctr.R1.fq -p fastuniq.ctr.R2.fq
fastuniq -i list2 -o fastuniq.ko.R1.fq -p fastuniq.ko.R2.fq
grep '^@' fastuniq.ctr.R1.fq | sort | uniq | wc -l
22295210 乘以四后也是行數(shù)；
去重率：
ctrl:80.23%
ko:86.58%
比對：
bowtie2 -p 20 -x $index -1 fastuniq.ctr.R1.fq -2 fastuniq.ctr.R2.fq -S fastuniq.ctr.sam?&&
bowtie2 -p 20 -x $index -1?fastuniq.ko.R1.fq?-2?fastuniq.ko.R2.fq?-S?fastuniq.ko.sam
比對結(jié)果：
查看sam：
44590443 fastuniq.ctr.sam
44155981 fastuniq.ko.sam
igv:
samtools sort --threads 10 -m 2G -o fastuniq.ctr.bam fastuniq.ctr.sam &&?
samtools sort --threads 10 -m 2G -o fastuniq.ko.bam fastuniq.ko.sam &&
samtools index?fastuniq.ctr.bam &&
samtools index??fastuniq.ko.bam
提取mapped：
samtools view -h -@ 10 -F 4?fastuniq.ctr.sam >?fastuniq.ctr.mapped.sam &&
samtools view -h -@ 10 -F 4?fastuniq.ko.sam >?fastuniq.ko.mapped.sam
查看mapped行數(shù)：
提取mapped后不能用igv秩铆。原因是缺少header：
解決：提取mapped時加-h參數(shù)：ref：[E::sam_parse1] missing SAM header
2.seqkit：
網(wǎng)站：https://bioinf.shenwei.me/seqkit/
用法：
命令：
seqkit rmdup -s ctr.R1.fq -o seqkit.ctr.R1.fq.gz -d seqkit.ctr.R1.duplicated.fq.gz -D seqkit.ctr.R1.duplicated.txt -j 5
seqkit rmdup -s ctr.R2.fq -o seqkit.ctr.R2.fq.gz -d seqkit.ctr.R2.duplicated.fq.gz -D seqkit.ctr.R2.duplicated.txt -j 5
seqkit rmdup -s ko.R1.fq -o seqkit.ko.R1.fq.gz -d seqkit.ko.R1.duplicated.fq.gz -D seqkit.ko.R1.duplicated.txt -j 5
seqkit rmdup -s ko.R2.fq -o seqkit.ko.R2.fq.gz -d seqkit.ko.R2.duplicated.fq.gz -D seqkit.ko.R2.duplicated.txt -j 5
查看行數(shù)：
zcat seqkit.ctr.R1.fq.gz | wc -l -> 57594872 51.82% [去重]
zcat seqkit.ctr.R2.fq.gz | wc -l -> 63321348 56.97%??[去重]
zcat seqkit.ko.R1.fq.gz | wc -l -> 60717652 54.63%??[去重]
zcat seqkit.ko.R2.fq.gz | wc -l -> 67585076 66.26%??[去重]
比對：
bowtie2 -p 20 -x $index -1?seqkit.ctr.R1.fq.gz?-2?seqkit.ctr.R2.fq.gz?-S?seqkit.ctr.sam?&&
bowtie2 -p 20 -x $index -1?seqkit.ko.R1.fq?-2?seqkit.ko.R2.fq?-S?seqkit.ko.sam
報錯了：雙端不對齊
結(jié)論：
seqkit去重率約50-70%榛做，但是使用后雙端不齊浅乔，無法比對赌厅。但是可以保留去重掉的fastq焕刮。
3.super_deduper:
官網(wǎng)：https://github.com/dstreett/Super-Deduper
新地址：https://github.com/ibest/HTStream
用法：
命令：
super_deduper -1 ctr.R1.fq -2?ctr.R1.fq -g -p super_deduper.ctrl
super_deduper -1 ko.R1.fq -2?ko.R1.fq?-g -p?super_deduper.ko
查看行數(shù)：
zcat super_deduper.ctrl_R1.fastq.gz |wc -l -> 3915060 3.52% [去重]
zcat super_deduper.ctrl_R2.fastq.gz |wc -l -> 3915060 3.52% [去重]
zcat super_deduper.ko_R1.fastq.gz |wc -l -> 3971360 3.89%?[去重]
zcat super_deduper.ko_R2.fastq.gz |wc -l -> 3971360 3.89%?[去重]
修改參數(shù)：
super_deduper -1 ctr.R1.fq -2 ctr.R1.fq -g -p super_deduper.ctrl -q 30
super_deduper -1 ko.R1.fq -2 ko.R1.fq -g -p super_deduper.ko -q 20
查看again：
zcat super_deduper.ctrl_R1.fastq.gz | wc -l -> 3915060?3.52% [去重]
zcat super_deduper.ctrl_R2.fastq.gz | wc -l -> 3915060?
zcat super_deduper.ko_R1.fastq.gz | wc -l?-> 3971360?3.89%?[去重]
zcat super_deduper.ko_R2.fastq.gz | wc -l?-> 3971360? 改了參數(shù)后竟然沒變
比對：
bowtie2 -p 20 -x $index -1?super_deduper.ctrl_R1.fastq.gz?-2?super_deduper.ctrl_R2.fastq.gz?-S?super_deduper.ctrl.sam?&&
bowtie2 -p 20 -x $index -1?super_deduper.ko_R1.fastq.gz?-2?super_deduper.ko_R2.fastq.gz?-S?super_deduper.ko.sam
比對結(jié)果：
查看sam行數(shù)：
igv:
samtools sort --threads 10 -m 2G -o super_deduper.ctrl.bam super_deduper.ctrl.sam && samtools sort --threads 10 -m 2G -o super_deduper.ko.bam super_deduper.ko.sam
samtools index?super_deduper.ctrl.bam &&
samtools index??super_deduper.ko.bam
提取mapped：
samtools view -h -@ 10 -F 4?super_deduper.ctrl.sam >?super_deduper.ctrl.mapped.sam &&
samtools view -h -@ 10 -F 4?super_deduper.ko.sam >?super_deduper.ko.mapped.sam
查看mapped的行數(shù)
結(jié)論：
super_deduper率約3-4%注益，去重去的太多碴巾。原理是什么呢？
4.fastp:
官網(wǎng)：https://github.com/OpenGene/fastp
用法：
命令：
fastp -i ctr.R1.fq -I ctr.R2.fq -o fastp.ctr.R1.fq -O fastp.ctr.R2.fq
fastp -i ko.R1.fq -I ko.R2.fq -o fastp.ko.R1.fq -O fastp.ko.R2.fq
查看：
查看：
fastp.ctr.R1.fq ->?27082408*4 97.46%?[去重]
fastp.ctr.R2.fq?->?27082408*4?97.46%?[去重]
fastp.ko.R1.fq?->?24873179*4 97.54% [去重]
fastp.ko.R2.fq?->?24873179*4?97.54%?[去重]
比對：
bowtie2 -p 20 -x $index -1?fastp.ctr.R1.fq?-2?fastp.ctr.R2.fq?-S?fastp.ctr.sam?&&
bowtie2 -p 20 -x $index -1?fastp.ko.R1.fq?-2?fastp.ko.R2.fq?-S?fastp.ko.sam
比對情況：
提取mapped：
samtools view -h -@ 10 -F 4?fastp.ctr.sam >?fastp.ctr.mapped.sam &&
samtools view -h -@ 10 -F 4?fastp.ko.sam >?fastp.ko.mapped.sam
查看行：
35,942,791 fastp.ctr.mapped.sam
32,536,623 fastp.ko.mapped.sam
igv:
samtools sort --threads 10 -m 2G -o?fastp.ctr.mapped.bam?fastp.ctr.mapped.sam&& samtools sort --threads 10 -m 2G -o?fastp.ko.mapped.bamfastp.ko.mapped.sam &&
samtools index?fastp.ctr.mapped.bam??&&
samtools index?fastp.ko.mapped.bam
5.samtools：
官網(wǎng)：http://www.htslib.org/
samtools sort --threads 10 -n? ctr.bam?-o ctr.sort.bam &&
samtools fixmate?--threads 10 -m ctr.sort.bam ctr.fixmate.bam &&
samtools sort?--threads 10 ctr.fixmate.bam?-o ctr.positionsort.bam &&
samtools markdup --threads 10 -r ctr.positionsort.bam ctr.markdup.bam
samtools sort --threads 10 -n??ko.bam?-o?ko.sort.bam &&
samtools fixmate?--threads 10?-m?ko.sort.bam?ko.fixmate.bam &&
samtools sort?--threads 10?ko.fixmate.bam?-o?ko.positionsort.bam &&
samtools markdup?--threads 10?-r?ko.positionsort.bam ko.markdup.bam?
查看行：
samtools view??ctr.markdup.bam | wc -l?39,334,011
samtools?view ko.markdup.bam | wc -l?39,170,695
提取mapped：
samtools view -h -@ 10 -F 4??ctr.markdup.bam > ctr.markdup.mapped.sam &&
samtools view -h -@ 10 -F 4??ko.markdup.bam >?ko.markdup.mapped.sam
查看mapped的行數(shù)：
wc -l?ctr.markdup.mapped.sam? ->?20,069,023
wc -l?ko.markdup.mapped.sam ->?21,054,225
igv:
samtools sort --threads 10 -m 2G -o ctr.markdup.mapped.bam ctr.markdup.mapped.sam && samtools sort --threads 10 -m 2G -o ko.markdup.mapped.bam ko.markdup.mapped.sam &&
samtools index?ctr.markdup.mapped.bam &&
samtools index??ko.markdup.mapped.bam
6.picard:
官網(wǎng)：https://broadinstitute.github.io/picard/
用法：
直接刪除冗余：
java -jar /home/pc/biosoft/picard.jar MarkDuplicates REMOVE_DUPLICATES=true I=ctr.sort.bam O=picard.ctr.sorted.markdup.bam M=picard.ctr.markdup.txt
****************************************************
java -jar /home/pc/biosoft/picard.jar MarkDuplicates REMOVE_DUPLICATES=true I=ko.sort.bam O=picard.ko.sorted.markdup.bam M=picard.ko.markdup.txt
查看行：
samtools?view picard.ctr.sorted.markdup.bam | wc -l ->?39,302,611
samtools?view picard.ko.sorted.markdup.bam | wc -l ->?39,151,069
igv:
samtools index picard.ctr.sorted.markdup.bam
samtools index picard.ko.sorted.markdup.bam
提取mapped：
samtools view -h -@ 10 -F 4??picard.ctr.sorted.markdup.bam >?picard.ctr.sorted.markdup.mapped.sam &&?
samtools view -h -@ 10 -F 4??picard.ko.sorted.markdup.bam >?picard.ko.sorted.markdup.mapped.sam
查看行數(shù)：
20,037,624 picard.ctr.sorted.markdup.mapped.sam
21,034,600 picard.ko.sorted.markdup.mapped.sam
igv:
samtools sort --threads 10 -m 2G -o?picard.ctr.sorted.markdup.mapped.bam?picard.ctr.sorted.markdup.mapped.sam?&&
samtools sort --threads 10 -m 2G -o?picard.ko.sorted.markdup.mapped.bam?picard.ko.sorted.markdup.mapped.sam &&
samtools index?picard.ctr.sorted.markdup.mapped.bam?&&
samtools index?picard.ko.sorted.markdup.mapped.bam
# 比對效率低丑搔？
用hisat2比對看竟然比對效率提高這么多
