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> docker search 亿速云 #搜索组学大讲堂提供的所有镜像 NAME DESCRIPTION STARS OFFICIAL AUTOMATED 亿速云/gene-family gene-family analysis docker image 5 亿速云/rnaseq RNA-seq analysis docker image build by omics… 3 亿速云/gsds-v2 GSDS 2.0 – Gene Structure Display Server 1 亿速云/reseq whole genome resequence analysis 1 亿速云/biocontainer-base Biocontainers base Image centos7 1 亿速云/blast-plus blast+ v2.9.0 0 亿速云/isoseq3 isoseq3 v3.3.0 build by 亿速云 0 亿速云/bwa BWA v0.7.17 build by 亿速云 0 亿速云/blastall legacy blastall v2.2.26 0 亿速云/sratoolkit SRAtoolkit v2.10.3 and aspera v3.9.9.177872 0 亿速云/ampliseq-q2 Amplicon sequencing qiime2 v2020.2 image 0 亿速云/ampliseq-q1 Amplicon sequencing qiime1 v1.9.1 image 0 亿速云/samtools samtools v1.10 build by 亿速云 0 亿速云/bsaseq NGS Bulk Segregant Analysis image 0 亿速云/gwas gwas analysis images 0 > docker pull 亿速云/ampliseq-q1 #下载扩增子镜像 >docker run --rm -it -m 4G --cpus 1 -v D:\qiime1-16s:/work 亿速云/ampliseq-q1:latest #启动并进入镜像
qiime1 v1.9.1 mothur v.1.25.0 usearch 10.0.240 usearch71 6.1.544 vsearch v2.15.0
flash v2.15.0 序列合并 bioperl biopython fastqc multiqc fastp
blast-2.2.22 blat-36 cdhit-3.1 muscle-3.8.31 rdpclassifier-2.2 uclust
picrust-1.1.4 bugbase
ggplot2 Ternary DESeq2 edgeR ggtree vegan pheatmap
randomForest 机器学习 scikit-learn 机器学习 circos 圈图绘制 Krona 物种丰度圈图 LefSe 差异比较分析
注意:以上软件包的列表只是部分举例,实际安装的包还有更多。
fastmap.txt文件, 测序数据文件放在data文件夹中,物种注释数据库文件Greengene silva unite等放在database目录,需要这些测试数据的同学可以关注组学大讲堂公众号,并回复16s,即可得到;准备完成之后,目录结构如下:
[root@aefe86d682b1 13:58:49 /work/amplicon_demo]# tree data |-- ERR3975186_1.fastq.gz |-- ERR3975186_2.fastq.gz |-- ERR3975187_1.fastq.gz `-- ERR3975187_2.fastq.gz fastmap.txt
设置一些环境变量方便后续调用:
dbdir=/work/database workdir=/work/amplicon_demo datadir=$workdir/data fastmap=$workdir/fastmap.txt mkdir /work/tmp export TMPDIR=/work/tmp #防止临时目录存储 不够 ######################database #各大数据库地址: silva_16S_97_seq=$dbdir/SILVA_132_QIIME_release/rep_set/rep_set_16S_only/97/silva_132_97_16S.fna silva_16S_97_tax=$dbdir/SILVA_132_QIIME_release/taxonomy/16S_only/97/taxonomy_7_levels.txt greengene_16S_97_seq=$dbdir/gg_13_8_otus/rep_set/97_otus.fasta greengene_16S_97_tax=$dbdir/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt silva_18S_97_seq=$dbdir/SILVA_132_QIIME_release/rep_set/rep_set_18S_only/97/silva_132_97_18S.fna silva_18S_97_tax=$dbdir/SILVA_132_QIIME_release/taxonomy/18S_only/97/taxonomy_7_levels.txt unite_ITS_97_seq=$dbdir/unite_ITS8.2/sh_refs_qiime_ver8_97_04.02.2020.fasta unite_ITS_97_tax=$dbdir/unite_ITS8.2/sh_taxonomy_qiime_ver8_97_04.02.2020.txt
cd $workdir #回到工作目录 mkdir 1.merge_pe for i in `cat $fastmap |grep -v '#'|cut -f 1` ;do echo "RUN CMD: flash $datadir/${i}_1.fastq.gz $datadir/${i}_2.fastq.gz \ -m 10 -x 0.2 -p 33 -t 1 \ -o $i -d 1.merge_pe" flash $datadir/${i}_1.fastq.gz $datadir/${i}_2.fastq.gz \ -m 10 -x 0.2 -p 33 -t 1 \ -o $i -d 1.merge_pe done
cd $workdir #回到工作目录 mkdir 2.fastqc #fastqc查看数据质量分布等 fastqc -t 2 $workdir/1.merge_pe/*extendedFrags.fastq -o $workdir/2.fastqc #质控结果汇总 cd $workdir/2.fastqc multiqc .
4.数据质控:对原始序列进行去接头,引物,删除低质量的reads等等
cd $workdir #回到工作目录 mkdir 3.data_qc cd 3.data_qc #利用fastp工具去除adapter #--qualified_quality_phred the quality value that a base is qualified. # Default 15 means phred quality >=Q15 is qualified. (int [=15]) #--unqualified_percent_limit how many percents of bases are allowed to be unqualified #--n_base_limit if one read's number of N base is >n_base_limit, # then this read/pair is discarded #--detect_adapter_for_pe 接头序列未知 可设置软件自动识别常见接头 # for i in `cat $fastmap |grep -v '#'|cut -f 1`; do echo "RUN CMD: fastp --thread 1 --qualified_quality_phred 10 \ --unqualified_percent_limit 50 \ --n_base_limit 10 \ --length_required 300 \ --trim_front1 29 \ --trim_tail1 18 \ -i $workdir/1.merge_pe/${i}.extendedFrags.fastq \ -o ${i}.clean_tags.fq.gz \ --adapter_fasta $workdir/data/illumina_multiplex.fa -h ${i}.html -j ${i}.json" fastp --thread 1 --qualified_quality_phred 10 \ --unqualified_percent_limit 50 \ --n_base_limit 10 \ --length_required 300 \ --trim_front1 29 \ --trim_tail1 18 \ -i $workdir/1.merge_pe/${i}.extendedFrags.fastq \ -o ${i}.clean_tags.fq.gz \ --detect_adapter_for_pe -h ${i}.html -j ${i}.json done
cd $workdir #回到工作目录mkdir 4.remove_chimerascd 4.remove_chimeras #去除嵌合体 for i in `cat $fastmap |grep -v '#'|cut -f 1`; do #相同重复序列合并 vsearch --derep_fulllength $workdir/3.data_qc/${i}.clean_tags.fq.gz \ --sizeout --output ${i}.derep.fa #去嵌合体 vsearch --uchime3_deno ${i}.derep.fa \ --sizein --sizeout \ --nonchimeras ${i}.denovo.nonchimeras.rep.fa #相同序列还原为多个 vsearch --rereplicate ${i}.denovo.nonchimeras.rep.fa --output ${i}.denovo.nonchimeras.fadone #根据参考序列去除嵌合体for i in `cat $fastmap |grep -v '#'|cut -f 1`; do vsearch --uchime_ref ${i}.denovo.nonchimeras.fa \ --db $dbdir/rdp_gold.fa \ --sizein --sizeout --fasta_width 0 \ --nonchimeras ${i}.ref.nonchimeras.fadone
cd $workdir #回到工作目录 mkdir 5.pick_otu_qiime cd 5.pick_otu_qiime #合并fasta文件,并加序列号 for i in `cat $fastmap |grep -v '#'|cut -f 1`; do rename_fa_id.pl -f $workdir/4.remove_chimeras/$i.ref.nonchimeras.fa \ -n $i -out $i.fa done #合并fa文件到qiime.fasta 之后删除所有单个样本的fa文件 cat *fa >qiime.fasta rm -f *fa ###方法1:pick_de_novo_otus.py ###输出qiime pick otu 参数,更多:http://qiime.org/scripts/pick_otus.html echo pick_otus:denovo_otu_id_prefix OTU >> otu_params_de_novo.txt echo pick_otus:similarity 0.97 >> otu_params_de_novo.txt echo pick_otus:otu_picking_method uclust >> otu_params_de_novo.txt #sortmerna, mothur, trie, uclust_ref, usearch, usearch_ref, blast, usearch71, usearch71_ref,sumaclust, swarm, prefix_suffix, cdhit, uclust. echo assign_taxonomy:reference_seqs_fp $silva_16S_97_seq >> otu_params_de_novo.txt echo assign_taxonomy:id_to_taxonomy_fp $silva_16S_97_tax >> otu_params_de_novo.txt echo assign_taxonomy:similarity 0.8 >>otu_params_de_novo.txt echo assign_taxonomy:assignment_method uclust >>otu_params_de_novo.txt # rdp, blast,rtax, mothur, uclust, sortmerna如果是ITS/18S数据,建议数据库更改为UNITE,方法改为blast。详细使用说明,请读官方文档:http://qiime.org/scripts/assign_taxonomy.html pick_de_novo_otus.py -i qiime.fasta -f -o pick_de_novo_otus -p otu_params_de_novo.txt
cd $workdir #回到工作目录 mkdir 8.alpha_diversity cd 8.alpha_diversity #alpha多样性指数展示 biom summarize-table -i $workdir/5.pick_otu_qiime/pick_de_novo_otus/otu_table_clean_rare.biom echo alpha_diversity:metrics observed_species,PD_whole_tree,shannon,chao1,simpson,goods_coverage > alpha_params.txt alpha_rarefaction.py -f -i $workdir/5.pick_otu_qiime/pick_de_novo_otus/otu_table_clean.biom -m $fastmap -o ./ -p alpha_params.txt -t $workdir/5.pick_otu_qiime/pick_de_novo_otus/rep_set.tre --retain_intermediate_files --min_rare_depth 40 --max_rare_depth 2032 --num_steps 10 #多样性指数差异比较qiime 自带检验与绘图 compare_alpha_diversity.py \ -i alpha_div_collated/chao1.txt \ -o alpha_chao1_stats \ -m $fastmap \ -t nonparametric \ -c city compare_alpha_diversity.py \ -i alpha_div_collated/chao1.txt \ -o alpha_chao1_stats \ -m $fastmap \ -t parametric \ -c city
cd $workdir #回到工作目录 mkdir 9.beta_diversity cd 9.beta_diversity echo beta_diversity:metrics binary_jaccard,bray_curtis,unweighted_unifrac,weighted_unifrac,binary_euclidean > beta_params.txt #-e 设置抽平数 beta_diversity_through_plots.py -f -i $workdir/5.pick_otu_qiime/pick_de_novo_otus/otu_table_clean.biom -m $fastmap -o ./ -t $workdir/5.pick_otu_qiime/pick_de_novo_otus/rep_set.tre -e 2844 -p beta_params.txt #beta多样性adonis检验 compare_categories.py --method adonis -i unweighted_unifrac_dm.txt -m $fastmap -c Treatment -o adonis_out -n 999
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