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> docker search 億速云 #搜索組學大講堂提供的所有鏡像 NAME DESCRIPTION STARS OFFICIAL AUTOMATED 億速云/gene-family gene-family analysis docker image 5 億速云/rnaseq RNA-seq analysis docker image build by omics… 3 億速云/gsds-v2 GSDS 2.0 – Gene Structure Display Server 1 億速云/reseq whole genome resequence analysis 1 億速云/biocontainer-base Biocontainers base Image centos7 1 億速云/blast-plus blast+ v2.9.0 0 億速云/isoseq3 isoseq3 v3.3.0 build by 億速云 0 億速云/bwa BWA v0.7.17 build by 億速云 0 億速云/blastall legacy blastall v2.2.26 0 億速云/sratoolkit SRAtoolkit v2.10.3 and aspera v3.9.9.177872 0 億速云/ampliseq-q2 Amplicon sequencing qiime2 v2020.2 image 0 億速云/ampliseq-q1 Amplicon sequencing qiime1 v1.9.1 image 0 億速云/samtools samtools v1.10 build by 億速云 0 億速云/bsaseq NGS Bulk Segregant Analysis image 0 億速云/gwas gwas analysis images 0 > docker pull 億速云/ampliseq-q1 #下載擴增子鏡像 >docker run --rm -it -m 4G --cpus 1 -v D:\qiime1-16s:/work 億速云/ampliseq-q1:latest #啟動并進入鏡像
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 機器學習 scikit-learn 機器學習 circos 圈圖繪制 Krona 物種豐度圈圖 LefSe 差異比較分析
注意:以上軟件包的列表只是部分舉例,實際安裝的包還有更多。
fastmap.txt文件, 測序數據文件放在data文件夾中,物種注釋數據庫文件Greengene silva unite等放在database目錄,需要這些測試數據的同學可以關注組學大講堂公眾號,并回復16s,即可得到;準備完成之后,目錄結構如下:
[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
設置一些環境變量方便后續調用:
dbdir=/work/database workdir=/work/amplicon_demo datadir=$workdir/data fastmap=$workdir/fastmap.txt mkdir /work/tmp export TMPDIR=/work/tmp #防止臨時目錄存儲 不夠 ######################database #各大數據庫地址: 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 #回到工作目錄
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
donecd $workdir #回到工作目錄 mkdir 2.fastqc #fastqc查看數據質量分布等 fastqc -t 2 $workdir/1.merge_pe/*extendedFrags.fastq -o $workdir/2.fastqc #質控結果匯總 cd $workdir/2.fastqc multiqc .
4.數據質控:對原始序列進行去接頭,引物,刪除低質量的reads等等
cd $workdir #回到工作目錄
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 接頭序列未知 可設置軟件自動識別常見接頭
#
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
donecd $workdir #回到工作目錄mkdir 4.remove_chimerascd 4.remove_chimeras
#去除嵌合體
for i in `cat $fastmap |grep -v '#'|cut -f 1`; do #相同重復序列合并 vsearch --derep_fulllength $workdir/3.data_qc/${i}.clean_tags.fq.gz \ --sizeout --output ${i}.derep.fa #去嵌合體 vsearch --uchime3_deno ${i}.derep.fa \ --sizein --sizeout \ --nonchimeras ${i}.denovo.nonchimeras.rep.fa #相同序列還原為多個 vsearch --rereplicate ${i}.denovo.nonchimeras.rep.fa --output ${i}.denovo.nonchimeras.fadone
#根據參考序列去除嵌合體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.fadonecd $workdir #回到工作目錄 mkdir 5.pick_otu_qiime cd 5.pick_otu_qiime #合并fasta文件,并加序列號 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 之后刪除所有單個樣本的fa文件 cat *fa >qiime.fasta rm -f *fa ###方法1:pick_de_novo_otus.py ###輸出qiime pick otu 參數,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數據,建議數據庫更改為UNITE,方法改為blast。詳細使用說明,請讀官方文檔: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 #回到工作目錄 mkdir 8.alpha_diversity cd 8.alpha_diversity #alpha多樣性指數展示 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 #多樣性指數差異比較qiime 自帶檢驗與繪圖 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 #回到工作目錄 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 設置抽平數 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多樣性adonis檢驗 compare_categories.py --method adonis -i unweighted_unifrac_dm.txt -m $fastmap -c Treatment -o adonis_out -n 999
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