############ ###Stacks### ############ #first create folder inside your RADseq working directory mkdir raw barcodes samples stacks stackscor #create a file containing the barcodes and put it in /barcodes directory #barcodes ATGTGTCGCCAA TCTGAGCGTACA GATCTGAAGCTC CGACGATACTTG CTAGATGCTGAC GACACCGTATGT ##de novo mapping pipeline: ##### #process_radtags #s_T_1_sequence.txt = concatened first read files [from 3 lanes] #s_T_2_sequence.txt = concatened second read files [from 3 lanes] process_radtags -1 ./raw/s_T_1_sequence.txt -2 ./raw/s_T_2_sequence.txt -o ./samples/ \ -b ./barcodes -e ecoRI -c -q -r -i fastq -D -E phred64 \ --adapter_2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT \ --adapter_mm 2 ##### ##### #ustacks script #!/bin/bash files='ATGTGTCGCCAA TCTGAGCGTACA GATCTGAAGCTC CGACGATACTTG CTAGATGCTGAC GACACCGTATGT' #create individual catalog loci i=1 for i in $files do ustacks -t fastq -f ./samples/sample_{i}.1.fq -o ./stacks -i 1 -m 3 \ -M 2 -p 15 -r -d --max_locus_stacks 5 ##### ##### #cstacks script #!/bin/bash files='ATGTGTCGCCAA TCTGAGCGTACA GATCTGAAGCTC CGACGATACTTG CTAGATGCTGAC GACACCGTATGT' samp="" for i in $files do samp+="-s ./stacks/sample_{i}.1"; done # #Rebuild the catalog. # cstacks -b 1 -p 15 -n 3 $samp -o ./stacks --report_mmatches ##### ##### #sstacks script #!/bin/bash files='ATGTGTCGCCAA TCTGAGCGTACA GATCTGAAGCTC CGACGATACTTG CTAGATGCTGAC GACACCGTATGT' # #Match samples against catalog stacks pipeline # i=1 for i in $files do sstacks -b 1 -c ./stacks/batch_1 -s ./stacks/sample_{i}.1 -o ./stacks let "i+=1"; done ##### ##### #rxstacks pipeline/script #!/bin/bash files="sample_ATGTGTCGCCAA.1 sample_TCTGAGCGTACA.1 sample_GATCTGAAGCTC.1 sample_CGACGATACTTG.1 sample_CTAGATGCTGAC.1 sample_GACACCGTATGT.1" # #Make population-based corrections. # rxstacks -b 1 -P ./stacks/ -o ./stackscor/ \ --lnl_lim -10.0 --lnl_dist -t 8 \ --conf_lim 0.25 --prune_haplo \ --model_type snp --alpha 0.10 \ --verbose samp="" for file in $files do samp+="-s ./stackscor/$file "; done # #Rebuild the catalog. # cstacks -b 1 -n 3 -p 15 -o ./stackscor $samp --report_mmatches # #Rematch the catalog. # for file in $files do sstacks -b 1 -c ./stackscor/batch_1 -s ./stackscor/$file -o ./stackscor done ##### ##### #populations script populations -b 1 -P ./stackscor/ -s -t 15 -m 5 --lnl_lim -10 --vcf ##### ##### #Aditional steps to generate whitelist containing final SNP set #It was necessary to assembly minicontigs only associated with the final SNP set #export based on whitelist (final snp filtering) populations -b 1 -P ./stackscor/ -s -t 15 -W ./whitelistN3.tsv --vcf --fasta #sort read pairs based whitelist stackscor N ind >= 3 sort_read_pairs.pl -p ./stackscor -s ./samples/ -o ./pairsWhite3N/-t fasta -d -w whitelistN3.tsv #exec_velvet.pl #kmer27 whitelist exec_velvet.pl -s ./pairsWhite3N/ -o ./assembled/pairsWList/ -c -H 27 -M 200 -L -e /usr/local/bin ###### ########## ###Bash### ########## #Manual filtering using Linux shell ###### ################## ###RepeatMasker### ################## #Identify and annotate repetitive elements based on similarity RepeatMasker -species Arthropoda ###### ########## ###MiSA### ########## #Microsatellite search misa.pl #misa.ini #paramenters file definition(unit_size,min_repeats): 2-6 3-5 4-5 5-5 6-5 interruptions(max_difference_between_2_SSRs): 100 ###### ######################### ###Command-line blastx### ######################### blastx -query -db nr -out -outfmt 5 #outfmt means the output in .xml (you need this to input in Blast2GO