Get 2bRAD scripts:
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#download the scripts from github
cdh
git clone https://github.com/z0on/2bRAD_denovo.git
#add the path to 2bRAD to your PATH variable
cd 2bRAD_denovo
2bRAD_PATH=$(pwd)
export PATH="$PATH:~/2bRAD_denovo/"
#test you have access
cds
vcf2map.pl -h
#if you get a help menus it worked |
Check the reads
We sequenced four different samples with single-end reads across two lanes on an Illumina Hiseq.
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#In the 2bRAD library prep the adapters can be ligated to the fragment in either
#fragment in either orientation (note the mirrored sticky ends left by bcgl).
#So the genomic fragment may have been read from either direction
#check for reverse complement of cut site
grep "GCA......TCG" A_cat.fastq | wc -l
grep "GCA......TCG" B_cat.fastq | wc -l
#201574
#202908
#(216159 + 201574)/426295 = 0.98
#(220686 + 202908)/444053 = 0.95 |
Separate the pooled fastq files based on the ligation barcodes found at the end of the reads.
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#The command to run trim2bRAD_2barcodes_dedup.pl is complicated, so
#another script -- 2bRAD_trim_launch_dedup.pl -- is used to generate the command for us:
2bRAD_trim_launch_dedup.pl cat.fastq > dedupCommands
#look at the commands file
cat dedupCommands
#returns our next commands to execute:
trim2bRAD_2barcodes_dedup.pl input=A_CTGCAG_R1.fastq site=".{12}CGA.{6}TGC.{12}|.{12}GCA.{6}TCG.{12}" adaptor="AGATC" sampleID=100
trim2bRAD_2barcodes_dedup.pl input=B_GAAGTT_R1.fastq site=".{12}CGA.{6}TGC.{12}|.{12}GCA.{6}TCG.{12}" adaptor="AGATC" sampleID=100 |
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#search for a line in the barcode file with both the barcodes
grep TGGT barcode_data.tsv | grep GAAGTT
#MCNA4
#Why search for TGGT instead of ACCA?
#The ligation barcode on 3Illbc was TGGT, but was then read as ACCA.
#When recording which barcode is which, easier to record the antiBC sequence
#(see 2bRAD library preparation protocol):
https://github.com/z0on/2bRAD_denovo/blob/master/2bRAD_protocol_june1_2018.pdf
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Complete.