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Kneaddata, Fastqc, Trimmomatic,

Kneaddata, Fastqc, Trimmomatic,

作者: 胡童远 | 来源:发表于2021-02-04 17:54 被阅读0次

    kneaddata的下载,安装,使用:
    宏转录组分析:kneaddata质控、SortMeRNA去rRNA

    1 FastQC


    主页:

    http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

    下载页:

    http://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc

    下载,安装:

    wget -c http://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.9.zip
    unzip fastqc_v0.11.9.zip
    cd FastQC
    chmod 755 fastqc
    fastqc --help
    

    使用:

    mkdir fastqc
    /hwfssz1/ST_META/PN/hutongyuan/software/FastQC/./fastqc SRR413773_1.fastq -o fastqc/
    /hwfssz1/ST_META/PN/hutongyuan/software/FastQC/./fastqc SRR413773_2.fastq -o fastqc/
    

    2 Trimmomatic

    主页,下载页:

    http://www.usadellab.org/cms/?page=trimmomatic

    下载,安装:

    wget -c http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/Trimmomatic-0.39.zip
    cd Trimmomatic-0.39
    java -jar trimmomatic-0.39.jar --help
    
    conda install kneaddata  # 自动安装trimmomatic
    conda activate env
    trimmomatic --help
    

    使用:

    time trimmomatic PE -phred33 -threads 40 \
    SRR1778451_1.fastq SRR1778451_2.fastq \
    trimmomatic/SRR1778451_1_paired.fastq \
    trimmomatic/SRR1778451_1_unpaired.fastq \
    trimmomatic/SRR1778451_2_paired.fastq \
    trimmomatic/SRR1778451_2_unpaired.fastq \
    SLIDINGWINDOW:4:20 MINLEN:50
    

    3 Bowtie2

    下载,安装:

    conda install bowtie2
    

    建库:

    bowtie2-build -f hg38.fasta hg38 --threads 16
    

    比对-单端:去宿主,取unmapped序列 速度极慢,不知为何?

    mkdir bowtie2
    
    bowtie2 -f -U SRR413773_1.fasta \
    -p 16 --quiet \
    -S bowtie2/SRR413773_1.sam \
    -x temple/TF01-11 \
    --al bowtie2/SRR413773_1.tsv
    
    rm bowtie2/SRR413773_1.sam
    

    比对-双端:去宿主,取unmapped序列

    time bowtie2 -q -p 40 --quiet \
    -1 trimmomatic/SRR1778451_1_paired.fastq \
    -2 trimmomatic/SRR1778451_2_paired.fastq \
    -S bowtie2/SRR1778451.sam \
    -x /public/home/zzumgg03/huty/databases/hg38/hg38 \
    --un-conc bowtie2/SRR1778451_bowtie2.fastq
    
    rm bowtie2/SRR1778451.sam
    

    参数:

    -q: fastq [标签]
    -f: fasta [标签]
    --phred33: 测序碱基的质量体系,现在基本都是33
    -p: 线程数
    --no-unal:不保留未必对上的记录
    -U: unpaired
    -1/-2: paired read1 read2
    -x: temple
    -S: output sam
    --al: unpaired aligned
    --un: unpaired unaligned
    --al-conc: paired aligned
    --un-conc: paired unaligned
    

    4 kneaddata

    下载,安装:默认同时安装trimmomatic bowtie2 fastqc

    conda install kneaddata
    kneaddata --help
    

    质检,修剪,去宿主:

    source /public/home/zzumgg03/huty/softwares/miniconda3/etc/profile.d/conda.sh
    conda activate r403
    time kneaddata -t 40 -v \
    -i rawdata/SRR_list_8/SRR1778450/SRR1778450_1.fastq \
    -i rawdata/SRR_list_8/SRR1778450/SRR1778450_2.fastq \
    -o result/kneaddata/SRR_list_8/ \
    --trimmomatic ~/huty/softwares/miniconda3/envs/r403/share/trimmomatic-0.39-1/ \
    --max-memory 80g \
    --trimmomatic-options "SLIDINGWINDOW:4:20 MINLEN:50" \
    -db /public/home/zzumgg03/huty/databases/hg38/ \
    --bowtie2-options "--very-sensitive --dovetail --reoeder" \
    --fastqc ~/huty/softwares/miniconda3/envs/r403/bin/ \
    --run-fastqc-start \
    --run-fastqc-end \
    --remove-intermediate-output
    

    参考:
    去污染(宿主)过程记录
    随机宏基因组测序数据质量控制和去宿主的分析流程和常见问题

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