2023-07-20 重测序数据比对

作者: 麦冬花儿 | 来源:发表于2023-08-05 21:23 被阅读0次

HISAT-StringTie-Ballgown流程的解释（原理
ATAC比对率低
重测序方法整理
BWA和Bowtie的安装使用及区别
重测序比对流程
从bam文件中提取未比对上的reads——so easy!
STAR 2-pass, picard 到gatk的使用
SAMtools使用小技巧
图片2022-04-20
DNA甲基化输出文件说明-BatMeth2

全局比对：同一物种整体评价
局部比对：不同物种局部评价

安装软件

# Installing Bowtie1 (http://bowtie-bio.sourceforge.net/index.shtml)
#wget https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.3.1/bowtie-1.3.1-linux-x86_64.zip -P ~/software
unzip ~/software/bowtie-1.3.1-linux-x86_64.zip -d /opt/biosoft/
echo 'PATH=$PATH:/opt/biosoft/bowtie-1.3.-linux-x86_64/' >> ~/.bashrc
source ~/.bashrc

# Installing bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
#wget https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.5.1/bowtie2-2.5.1-linux-x86_64.zip -P ~/software
unzip ~/software/bowtie2-2.5.1-linux-x86_64.zip -d /opt/biosoft/
echo 'PATH=$PATH:/opt/biosoft/bowtie2-2.5.1-linux-x86_64/' >> ~/.bashrc
source ~/.bashrc

比对

mkdir -p /home/train/06.reads_aligment
cd /home/train/06.reads_aligment

# Bowtie2 practise
mkdir -p /home/train/06.reads_aligment/bowtie2
cd /home/train/06.reads_aligment/bowtie2

ln -s ~/00.incipient_data/data_for_genome_assembling/assemblies_of_Malassezia_sympodialis/Malassezia_sympodialis.genome_V01.fasta genome.fasta
ln -s ~/03.sequencing_data_preprocessing/V?.?.fastq ./

bowtie2-build --threads 8 genome.fasta genome #建立索引
bowtie2 -p 8 -x genome -1 V1.1.fastq -2 V1.2.fastq -S V1.sam --rg-id V1 --rg "PL:Illumina" --rg "SM:V1" 2> V1.bowtie2.log
# real  1m26.553s
# user  11m12.705s
# sys   0m13.891s

bowtie2 -p 8 -x genome -1 V2.1.fastq -2 V2.2.fastq -S V2.sam --rg-id V2 --rg "PL:Illumina" --rg "SM:V2" 2> V2.bowtie2.log

[train@MiWiFi-R3P-srv bowtie2]$ cat V2.bowtie2.log 
2240991 reads; of these:
  2240991 (100.00%) were paired; of these:
    693164 (30.93%) aligned concordantly 0 times
    1459657 (65.13%) aligned concordantly exactly 1 time
    88170 (3.93%) aligned concordantly >1 times
    ----
    693164 pairs aligned concordantly 0 times; of these:
      32957 (4.75%) aligned discordantly 1 time
    ----
    660207 pairs aligned 0 times concordantly or discordantly; of these:
      1320414 mates make up the pairs; of these:
        1308702 (99.11%) aligned 0 times
        7570 (0.57%) aligned exactly 1 time
        4142 (0.31%) aligned >1 times
70.80% overall alignment rate # mappling rate较低是由于线粒体基因组的干扰

BWA
软件下载与安装

# Installing BWA (https://sourceforge.net/projects/bio-bwa/files/)
#wget https://sourceforge.net/projects/bio-bwa/files/bwa-0.7.17.tar.bz2 -P ~/software/
tar jxf ~/software/bwa-0.7.17.tar.bz2 -C /opt/biosoft/
cd /opt/biosoft/bwa-0.7.17/
make CC='gcc -fcommon' -j 4
echo 'PATH=$PATH:/opt/biosoft/bwa-0.7.17/' >> ~/.bashrc
source ~/.bashrc

# BWA practise
mkdir -p /home/train/06.reads_aligment/bwa
cd /home/train/06.reads_aligment/bwa

ln -s ~/00.incipient_data/data_for_genome_assembling/assemblies_of_Malassezia_sympodialis/Malassezia_sympodialis.genome_V01.fasta genome.fasta
ln -s ~/03.sequencing_data_preprocessing/V?.?.fastq ./

bwa index genome.fasta -p genome

bwa mem -t 8 -R '@RG\tID:V1\tSM:V1' genome V1.1.fastq V1.2.fastq > V1.mem.sam
# real  0m23.141s
# user  2m31.3.0s
# sys   0m3.354s

bwa bwasw -t 8 genome V1.1.fastq V1.2.fastq > V1.bwasw.sam
# real  2m31.854s
# user  18m43.008s
# sys   0m30.774s

bwa aln -t 8 genome V1.1.fastq > V1.1.sai
# real  0m9.566s
# user  1m2.180s
# sys   0m0.321s
bwa aln -t 8 genome V1.2.fastq > V1.2.sai
bwa sampe genome V1.1.sai V1.2.sai V1.1.fastq V1.2.fastq > V1.backtrack.sam
# real  0m24.595s
# user  0m20.893s
# sys   0m1.737s