扩增子分析

export PATH=/home/llt/miniconda2/bin:$PATH
source activate qiime1

1. 质控
2. 拼接 join_paired_ends.py -f 1_1.fq.gz -r 2_2.fq.gz -m fastq-join -o join
3. extract_barcodes.py -f join_paired/fastqjoin.join.fastq -c barcode_single_end --bc1_len 12 -o barcodes
4. split_libraries_fastq.py -i barcodes/reads.fastq -b barcodes/barcodes.fastq -o split -m mapping.txt -q 19

wget https://pypi.python.org/packages/source/s/setuptools/setuptools-19.2.zip
    unzip setuptools-19.2.zip
    cd setuptools-19.2
    python setup.py install

wget https://pypi.python.org/packages/16/e3/06b45eea35359833e7c6fac824b604f1551c2fc7ba0f2bd318d8dd883eb9/cutadapt-1.14.tar.gz 
 cutadapt -g CCGGACTACVSGGGTAT -a WTTCKCGMCTGSTGCRC -e 0.15 -m 300 --discard-untrimmed seqs.fna -o cut_seq.fna

usearch
sed 's/ .*/;/g;s/>.*/&&/g;s/;>/;barcodelabel=/g;s/_[0-9]*;$/;/g' cut_seq.fna > usearch_seq.fa

identify_chimeric_seqs.py -m usearch61 -i split/usearch_seq.fa -o chimeras --suppress_usearch61_ref 
filter_fasta.py -f split/usearch_seq.fa -o non_chimeras/non_chimeras_seq.fasta -s chimeras/chimeras.txt -n 

pick_otus.py -i non_chimeras/non_chimeras_seq.fasta -o pick_otus_default
pick_otus.py -i non_chimeras/non_chimeras_seq.fasta -m usearch --db /home/llt/database/gold.fa  --minsize 2 -o pick_otus_usrarch_ref_gold
pick_rep_set.py -i pick_otus_default/non_chimeras_seq_otus.txt -f non_chimeras/non_chimeras_seq.fasta -o pick_otus_default/rep_set.fna
pick_otus.py -i non_chimeras.fasta -m usearch -s 0.95 --word_length 64 --suppress_reference_chimera_detection --minsize 2 -o usearch_qf_results_no_ref_chim_detection/  # mv usearchXXX usearch 

assign_taxonomy.py -i rep_set.fna -r /home/llt/database/gg_13_8_otus/rep_set/99_otus.fasta -t /home/llt/database/gg_13_8_otus/taxonomy/99_otu_taxonomy.txt -m blast

make_otu_table.py -i non_chimeras_seq_otus.txt -t blast_assigned_taxonomy/rep_set_tax_assignments.txt -o otu_table.biom
biom convert -i otu_table.biom -o table.from_biom_w_consensuslineage.txt --to-tsv --header-key taxonomy --output-metadata-id "ConsensusLineage" --table-type="OTU table"

clustalo -i rep_set.fna -o rep_set_align.fa --seqtype=DNA --full --force --threads=4
filter_alignment.py -i rep_set_align.fa -o filter/
make_phylogeny.py -i rep_set_align_pfiltered.fasta -0 rep_set.tree

qiime > biom summarize-table -i otu_table.biom 
qiime > single_rarefaction.py -i otu_table.biom -o otu_table_rara.biom -d 1626
 alpha_diversity.py -i otu_table_rara.biom -o alpha.txt -t rep_set.tree -m shannon,chao1,observed_otus,PD_whole_tree