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Demultiplexing ddRAD
Demultiplexing ddRAD
Demultiplexing ddRAD data with Stacks exercise:
Check out the reads to make sure they make sense:
check out reads
#navigate to the exercise directory cd intro_to_rad_2018/demultiplexing/process_ddRAD_stacks #we have two fastq files: Lib01_R1.fastq and Lib01_R2.fastq ls *.fastq #These files have paired end reads (two separate reads from either end of the same DNA fragment) #Double-check that the files have the same number of reads. (note that fastq genreally files have 4 lines per read) expr $(cat Lib01_R1.fastq | wc -l) / 4 expr $(cat Lib01_R2.fastq | wc -l) / 4 #53042 #(Note these are for demo purposes and shorter than they should be) #Looking up nlaIII, we see it's cut site: # CATG' # 'GTAC #check if we see this cut site in the forward reads grep CATG Lib01_R1.fastq | wc -l #compare this with the total number of reads we got above #do these numbers make sense? #look at where in the reads the cut site is (may help to paste into a text editor and character search) grep CATG Lib01_R1.fastq | head -n 30
Based on the library preparation, we expect that our forward reads were cut with the restriction enzyme nlaIII (NLA3).
Check that the forward reads fit this expectation.
Hint: look up restriction site and use grep
process the reads using process_radtags (part of the Stacks package):
process tags
#make a directory to put the resulting sample fastq files into mkdir sample_fastqs #look at the documentation for process_radtags ./process_radtags -h #execute the command to process the rad data ./process_radtags -i 'fastq' -1 Lib01_R1.fastq -2 Lib01_R2.fastq -o ./sample_fastqs/ -b barcodes_Lib1.tsv --inline_index -e 'nlaIII' -r --disable_rad_check
Check the results:
check results
#how many barcode combinations did we have? #First look at the barcodes file: cat barcodes_Lib1.tsv #then count the lines cat barcodes_Lib1.tsv | wc -l #how r1 and r2 fastq files did we output (ignore the 'rem' files) ls sample_fastqs/*AGCGAC.1.fq ls sample_fastqs/*AGCGAC.1.fq ls sample_fastqs/*AGCGAC.1.fq | wc -l ls sample_fastqs/*AGCGAC.1.fq | wc -l #are the paired end files still the same length? cat sample_fastqs/sample_CATAT-AGCGAC.1.fq | wc -l cat sample_fastqs/sample_CATAT-AGCGAC.2.fq | wc -l