#Commands per each run

$ fastqc -o FastQC_raw/ --noextract R1_FW.fastq.gz R2_RV.fastq.gz

$ bbmap/bbduk.sh in=R1_FW.fastq.gz outm=FW_3N.fastq.gz restrictleft=15 k=12 literal=AAACTGCCGAAT,CTTAATCCAGGT,AATTGACAACTG,GCCTAATTCACT mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R1_FW.fastq.gz outm=FW_4N.fastq.gz restrictleft=16 k=12 literal=AATTCCGTGACG,GCATACAGTAAT,TCCGTCGAACGA,ACGAATCAACTA mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R1_FW.fastq.gz outm=FW_5N.fastq.gz restrictleft=17 k=12 literal=AGTATATCCTAC,TCCGCCTCAGAA,GCCAGTTCAGTA,AGCTCTTCAAAC mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R1_FW.fastq.gz outm=FW_6N.fastq.gz restrictleft=18 k=12 literal=CCGAAACCGACT,TTTAAGCGACCT,ATACAAACGCCT,ATGCGAGACAAA mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R2_RV.fastq.gz outm=RV_3N.fastq.gz restrictleft=15 k=12 literal=TAAGGCAGAGGT,AGATATGTAGAC,GCCTCGGCGCGA,TTGTTGACCAAT,TCAACATGTACT mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R2_RV.fastq.gz outm=RV_4N.fastq.gz restrictleft=16 k=12 literal=CGAATACGAACA,ACCTCAAATTAG,ATCCTGCCATTC,AAACTGTAAGGC,GCGCTGATTCAA mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R2_RV.fastq.gz outm=RV_5N.fastq.gz restrictleft=17 k=12 literal=CCTGCTACACTT,TTAATTTCTAAA,TATTCTTGACTA,CCTCTCACATGG,TTAACCTACAAT mm=f hdist=0 rcomp=f

$ bbmap/bbduk.sh in=R2_RV.fastq.gz outm=RV_6N.fastq.gz restrictleft=18 k=12 literal=GCCGCAGAATAT,AGCGGCATGATT,AGCAAGGAATAA,CTTGTAAATATA,AGCAAACCAGAC mm=f hdist=0 rcomp=f

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE FW_3N.fastq.gz FW_3N_nospacer.fastq.gz HEADCROP:3

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE FW_4N.fastq.gz FW_4N_nospacer.fastq.gz HEADCROP:4

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE FW_5N.fastq.gz FW_5N_nospacer.fastq.gz HEADCROP:5

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE FW_6N.fastq.gz FW_6N_nospacer.fastq.gz HEADCROP:6

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE RV_3N.fastq.gz RV_3N_nospacer.fastq.gz HEADCROP:3

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE RV_4N.fastq.gz RV_4N_nospacer.fastq.gz HEADCROP:4

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE RV_5N.fastq.gz RV_5N_nospacer.fastq.gz HEADCROP:5

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar SE RV_6N.fastq.gz RV_6N_nospacer.fastq.gz HEADCROP:6

$ zcat FW_3N_nospacer.fastq.gz FW_4N_nospacer.fastq.gz FW_5N_nospacer.fastq.gz FW_6N_nospacer.fastq.gz | gzip -c > FW_nospacer.fastq.gz

$ zcat RV_3N_nospacer.fastq.gz RV_4N_nospacer.fastq.gz RV_5N_nospacer.fastq.gz RV_6N_nospacer.fastq.gz | gzip -c > RV_nospacer.fastq.gz

$ bbmap/repair.sh in1=FW_nospacer.fastq.gz in2=RV_nospacer.fastq.gz out1=FW_repaired.fastq.gz out2=RV_repaired.fastq.gz

$ java -jar /Trimmomatic-0.33/trimmomatic-0.33.jar PE -baseout Trimmed.fastq.gz FW_repaired.fastq.gz RV_repaired.fastq.gz TRAILING:10 SLIDINGWINDOW:10:20 MINLEN:200 TOPHRED33

$ bbmap/bbmerge.sh in1=Trimmed_1P.fastq.gz in2=Trimmed_2P.fastq.gz out=Joined.fastq.gz minoverlap=30 mismatches=0 mininsert=400 mininsert0=400 minq=19 verystrict=t

$ bbmap/bbduk.sh ref=PhiX_AF176034.fasta in=Joined.fastq.gz hdist=1 out=Joined_noPhiX.fastq.gz

$ qiime_software/extract_barcodes.py -f Joined_noPhiX.fastq.gz -o Barcodes_qiime -c barcode_paired_stitched -l 12 -L 12 --rev_comp_bc2

$ qiime_software/count_seqs.py -i Barcodes_qiime/barcodes.fastq

$ qiime_software/count_seqs.py -i Barcodes_qiime/reads.fastq

$ qiime_software/split_libraries_fastq.py -i Barcodes_qiime/reads.fastq -o Demultiplex/ -m MapFile.txt -b Barcodes_qiime/barcodes.fastq --max_barcode_errors 0 -p 0.8 -q 19 -r 3 -n 0 --barcode_type 24

$ qiime_software/extract_seqs_by_sample_id.py -i Demultiplex/seqs.fna -m MapFile.txt -o Ctrl_seqs.fna -s "Project:ctrl"

$ qiime_software/identify_chimeric_seqs.py -i Ctrl_seqs.fna -o Ctrl_chimera/ -m usearch61 -r gold_db.fa

$ qiime_software/filter_fasta.py -f Crtl_seqs.fna -o Ctrl_seqs_nochimeras.fna -n -s Ctrl_chimera/chimeras.txt

$ qiime_software/extract_seqs_by_sample_id.py -i Demultiplex/seqs.fna -o EMERGE_seqs.fna -m MapFile.txt -s "Project:EMERGE"

$ qiime_software/identify_chimeric_seqs.py -i EMERGE_seqs.fna -o EMERGE_seqs_chimera/ -m usearch61 -r gold_db.fa

$ qiime_software/filter_fasta.py -f EMERGE_seqs.fna -o EMERGE_seqs_nochimeras.fna -n -s EMERGE_seqs_chimera/chimeras.txt

#Commands for all runs together

$ cat Run1_EMERGE_seqs_nochimera.fna Run2_EMERGE_seqs_nochimera.fna Run3_EMERGE_seqs_nochimeras.fna Run4_EMERGE_seqs_nochimeras.fna Run5_EMERGE_seqs_nochimeras.fna > EMERGE_all_seqs.fna

$ qiime_software/pick_open_reference_otus.py -i EMERGE_all_seqs.fna -p Parameters_open-ref-otu_all.txt -o EMERGE_openref_otu/ -r /qiime_software/Silva119_release/rep_set/97/Silva_119_rep_set97.fna -s 0.01 -a --jobs_to_start 16

$ qiime_software/filter_otus_from_otu_table.py -i otu_table_mc2_w_tax_no_pynast_failures.biom --min_count_fraction 0.0001 -o otu_table_mc2_w_tax_no_pynast_failures_001.biom

$ qiime_software/biom summarize-table -i otu_table_mc2_w_tax_no_pynast_failures_001.biom -o otu_table_mc2_w_tax_no_pynast_failures_001_sum.txt

$ qiime_software/biom summarize-table --qualitative -i otu_table_mc2_w_tax_no_pynast_failures_001.biom -o otu_table_mc2_w_tax_no_pynast_failures_001_qual-sum.txt

$ qiime_software/summarize_taxa_through_plots.py -i otu_table_mc2_w_tax_no_pynast_failures_001.biom -o EMERGE_taxa_plots_001/ -s

$ qiime_software/core_diversity_analyses.py -i otu_table_mc2_w_tax_no_pynast_failures_001.biom -m All_map.txt -p diversity_metrics.txt -o Core_analysis_001_2000/ -e 2000 -t rep_set.tre -a --jobs_to_start 16