R/robot_prep_16S.R

In econtijoch/Biomass-Workflow: Microbial Density Data Generation and Analysis Tools

#' Function to produce a Beckmann Robot-friendly file and table for diluting samples for 16S Sequencing
#' @param dataset an input dataset that includes sample volumes needed for pcr, water volume needed for pcr, dna concentrations, and sample location information. 
#' @param n_barcode_plates the number of barcode plates available (i.e. if you have 96 unique sequencing barcodes, this would be 1)
#' @return a table contianing the necessary information for the robot to handle the dilution, as well as some QC data for evaluating if there will be any errors. This will also print to terminal the names of the plates necessary (it is useful to have your plates named.)
#' @export
#'

robot_prep_16S <- function(dataset, n_barcode_plates) {
    
    sample_number <- nrow(dataset)
    total_number_of_plates_needed <- ceiling(sample_number/96)
    number_of_runs_needed <- ceiling(sample_number/(96 * n_barcode_plates))
    
    dataset$BarcodePlate <- ""
    dataset$BarcodeWell <- ""
    dataset$SequencingRun <- ""
    
    for (k in 1:number_of_runs_needed) {
        if (k < number_of_runs_needed) {
            plates_in_run <- n_barcode_plates
        } else {
            plates_in_run <- total_number_of_plates_needed - (k - 1) * n_barcode_plates
        }
        for (i in 1:plates_in_run) {
            for (j in 1:96) {
                entry <- ((i - 1) * 96) + j + (k - 1) * n_barcode_plates * 96
                if (entry <= sample_number) {
                  dataset[entry, "BarcodePlate"] <- paste("Plate", i, sep = "")
                  row <- (j - 1)%/%12
                  column <- j - (12 * (row))
                  row_id <- c("A", "B", "C", "D", "E", "F", "G", "H")
                  dataset[entry, "BarcodeWell"] <- paste(row_id[row + 1], sprintf("%02d", column), sep = "")
                  dataset[entry, "SequencingRun"] <- paste("16S_Run", k, sep = "")
                }
                
            }
            
        }
        
    }
    
    if ("X16S_possible" %in% colnames(dataset)) {
        if (sum(dataset[, "X16S_possible"]) < nrow(dataset)) {
            cat("WARNING: Running robot prep on samples that have not passed the '16S possible' check. Re-check data and filter if necessary.\n")
        }
    }
    
    robot_table <- dataset %>% dplyr::mutate(vol_needed_for_PCR = 400/dna_concentration, water_volume_up_PCR = 200 - vol_needed_for_PCR) %>% dplyr::select(BarcodeID, PlateID, SampleWell, vol_needed_for_PCR, water_volume_up_PCR, 
        dna_concentration, BarcodePlate, BarcodeWell, SequencingRun)
    robot_table$BarcodeID <- as.character(robot_table$BarcodeID)
    robot_table$PlateID <- as.character(robot_table$PlateID)
    robot_table$Warning <- ""
    robot_table$DNASource <- paste("Unnormalized", robot_table$PlateID, sep = "_")
    robot_table$StartingConc <- robot_table$dna_concentration
    robot_table$FinalConc <- 0
    
    for (i in 1:nrow(robot_table)) {
        if (robot_table[i, "dna_concentration"] < 1.5) {
            robot_table[i, "vol_needed_for_PCR"] = 40
            robot_table[i, "water_volume_up_PCR"] = 0
            robot_table[i, "Warning"] = "[WARNING] **DID NOT PASS 16S_possible check. Starting Concentration < 1.5: Transferred 40 uL + 0 uL Water/EB"
            robot_table[i, "FinalConc"] = robot_table[i, "StartingConc"]
        } else if (robot_table[i, "dna_concentration"] <= 2) {
            robot_table[i, "vol_needed_for_PCR"] = 40
            robot_table[i, "water_volume_up_PCR"] = 0
            robot_table[i, "Warning"] = "[WARNING] 2 >= Starting Concentration > 1.5: Transferred 40 uL + 0 uL Water/EB"
            robot_table[i, "FinalConc"] = robot_table[i, "StartingConc"]
        } else if (robot_table[i, "dna_concentration"] <= 20) {
            robot_table[i, "vol_needed_for_PCR"] = robot_table[i, "vol_needed_for_PCR"]/5
            robot_table[i, "water_volume_up_PCR"] = robot_table[i, "water_volume_up_PCR"]/5
            robot_table[i, "Warning"] = "20 >= Starting Concentration > 2: Transferred [DNAvol + Water/EB]= 40 uL"
            robot_table[i, "FinalConc"] = (robot_table[i, "StartingConc"] * robot_table[i, "vol_needed_for_PCR"])/40
        } else if (robot_table[i, "dna_concentration"] <= 100) {
            robot_table[i, "vol_needed_for_PCR"] = robot_table[i, "vol_needed_for_PCR"]/2
            robot_table[i, "water_volume_up_PCR"] = robot_table[i, "water_volume_up_PCR"]/2
            robot_table[i, "Warning"] = "100 >= Starting Concentration > 20: Transferred [DNAvol + Water/EB]= 100 uL"
            robot_table[i, "FinalConc"] = (robot_table[i, "StartingConc"] * robot_table[i, "vol_needed_for_PCR"])/100
        } else if (robot_table[i, "dna_concentration"] <= 400) {
            robot_table[i, "vol_needed_for_PCR"] = robot_table[i, "vol_needed_for_PCR"] * 3/4
            robot_table[i, "water_volume_up_PCR"] = robot_table[i, "water_volume_up_PCR"] * 3/4
            robot_table[i, "Warning"] = "400 >= Starting Concentration > 100: Transferred [DNAvol + Water/EB]= 150 uL"
            robot_table[i, "FinalConc"] = (robot_table[i, "StartingConc"] * robot_table[i, "vol_needed_for_PCR"])/150
        } else {
            robot_table[i, "vol_needed_for_PCR"] = 1
            robot_table[i, "water_volume_up_PCR"] = 99
            robot_table[i, "Warning"] = "[WARNING] Starting Concentration > 400: Transferred 1uL DNA + 199 uL Water/EB"
            robot_table[i, "FinalConc"] = robot_table[i, "StartingConc"]/200
        }
        
        robot_table$DNASourceWell <- unlist(lapply(robot_table$SampleWell, well_parser))
        
        robot_table$DNA_Vol <- plyr::round_any(robot_table$vol_needed_for_PCR, 0.1)
        robot_table$Water_Vol <- plyr::round_any(robot_table$water_volume_up_PCR, 0.1)
        robot_table$Destination <- paste("Normalized", robot_table$BarcodePlate, sep = "_")
        robot_table$DestinationWell <- unlist(lapply(robot_table$BarcodeWell, well_parser))
        robot_table$WaterSource <- "WaterSource"
        robot_table$WaterWell <- 1
        robot_table$NormalizedVolume <- robot_table$DNA_Vol + robot_table$Water_Vol
        
        output <- as.data.frame(dplyr::select(robot_table, WaterSource, WaterWell, DNASource, DNASourceWell, Destination, 
            DestinationWell, DNA_Vol, Water_Vol, Warning, BarcodeID, PlateID, SampleWell, SequencingRun, BarcodePlate, 
            BarcodeWell, NormalizedVolume, StartingConc, FinalConc))
        
    }
    output <- output %>% dplyr::arrange(SequencingRun, BarcodePlate, DestinationWell)
    
    cat("Names of plates needed for robot dilution: \n")
    cat(paste(unique(output$PlateID), collapse = "\n"))
    
    return(output)
    
    
}