Post-sequencing functional analysis of synthetic UPTAG DNA barcodes of S. cerevisiae heterozygous and homozygous mutants.
#Experimental design: For heterozygous deletion pool that represents Essential genes, there are 4 replicates of E_SH1009 (Treatment), 4 replicates of E_MMS (Positive control), 4 replicates of E_DMSO (Negative control). For homozygous deletion pool that represents Non-essential genes, there are 4 replicates of NON_SH1009 (Treatment), 4 replicates of NON_MMS (Positive control), 4 replicates of NON_DMSO (Negative control).
#First: Processing.pl #In Processing.pl script, the converted fasta file was the input file that processed to remove the header lines using substitution function (s//), resulting in total of 12,000,000 pure sequence reads. Each read contained 50 bp from 5' to 3'; 10 bp index tag, 18 bp common forward U1 region, 20 bp UPTAG unique barcode, and 2 bp from U2 common reverse region.
#Second: Demultipexing.pl #In Demultipexing.pl script, the total 12,000,000 sequence reads were de-multiplexed based on the index tags (~10 bp) using sort function (sort//) in order to assign each sequence read to its original sample. As a result of sorting function, 24 output files were extracted, each file represents one PCR product called with the same name of its forward primer, S3 Table.
#Third: String.pl #String.pl script was written for joining each demultiplexed sequence reads into a string which makes the next step (matching & counting) more faster. 24 input demultiplexed files were converted to 24 output string-files.
#Fort: Match_Count.pl #Match_Count_Heterozygous_Barcodes.pl and Match_Count_Homozygous_Barcodes.pl scripts. Each string replicate was run through independent match_count.pl script to save time and memory. In each script, the frequency of each unique DNA barcode that represents one mutant was counted and assigned to its mutant’s systematic name using regular expression (~//ig).
#Finally: Assembly.pl #Assebly_Heterozygous_Counts.pl script was created to bring all the counts for each mutant form 12 Counting-files of heterozygous profile while Assebly_Homozygous_Counts.pl script was created to bring all the counts for each mutant form 12 Counting-files of homozygous profile using hash function ($hash) where the key was the systematic name of the mutant and the values were the counts of that mutant throughout treatment (SH1009), positive control (MMS), and negative control (DMSO).