spacr 0.0.35__py3-none-any.whl → 0.0.61__py3-none-any.whl

spacr/sequencing.py

@@ -0,0 +1,1130 @@
+import os, re, time, math, subprocess
+import numpy as np
+import pandas as pd
+import matplotlib.pyplot as plt
+import seaborn as sns
+from Bio import pairwise2
+import statsmodels.api as sm
+import statsmodels.formula.api as smf
+from scipy.stats import gmean
+from difflib import SequenceMatcher
+
+def reverse_complement(dna_sequence):
+    complement_dict = {'A': 'T', 'T': 'A', 'C': 'G', 'G': 'C', 'N':'N'}
+    reverse_seq = dna_sequence[::-1]
+    reverse_complement_seq = ''.join([complement_dict[base] for base in reverse_seq])
+    return reverse_complement_seq
+
+def complement(dna_sequence):
+    complement_dict = {'A': 'T', 'T': 'A', 'C': 'G', 'G': 'C', 'N':'N'}
+    complement_seq = ''.join([complement_dict[base] for base in dna_sequence])
+    return complement_seq
+
+def file_len(fname):
+    p = subprocess.Popen(['wc', '-l', fname], stdout=subprocess.PIPE, stderr=subprocess.PIPE)
+    result, err = p.communicate()
+    if p.returncode != 0:
+        raise IOError(err)
+    return int(result.strip().split()[0])
+
+def generate_plate_heatmap(df, plate_number, variable, grouping, min_max):
+    if grouping == 'mean':
+        temp = df.groupby(['plate','row','col']).mean()[variable]
+    if grouping == 'sum':
+        temp = df.groupby(['plate','row','col']).sum()[variable]
+    if grouping == 'count':
+        temp = df.groupby(['plate','row','col']).count()[variable]
+    if grouping in ['mean', 'count', 'sum']:
+        temp = pd.DataFrame(temp)
+    if min_max == 'all':  
+        min_max=[np.min(temp[variable]),np.max(temp[variable])]   
+    if min_max == 'allq':
+        min_max = np.quantile(temp[variable], [0.2, 0.98])
+    plate = df[df['plate'] == plate_number]
+    plate = pd.DataFrame(plate)
+    if grouping == 'mean':
+        plate = plate.groupby(['plate','row','col']).mean()[variable]
+    if grouping == 'sum':
+        plate = plate.groupby(['plate','row','col']).sum()[variable]
+    if grouping == 'count':
+        plate = plate.groupby(['plate','row','col']).count()[variable]
+    if grouping not in ['mean', 'count', 'sum']:
+        plate = plate.groupby(['plate','row','col']).mean()[variable]
+    if min_max == 'plate':
+        min_max=[np.min(plate[variable]),np.max(plate[variable])]
+    plate = pd.DataFrame(plate)
+    plate = plate.reset_index()
+    if 'plate' in plate.columns:
+        plate = plate.drop(['plate'], axis=1)
+    pcol = [*range(1,28,1)]
+    prow = [*range(1,17,1)]
+    new_col = []
+    for v in pcol:
+        col = 'c'+str(v)
+        new_col.append(col)
+    new_col.remove('c15')
+    new_row = []
+    for v in prow:
+        ro = 'r'+str(v)
+        new_row.append(ro)
+    plate_map = pd.DataFrame(columns=new_col, index = new_row)
+    for index, row in plate.iterrows():
+        r = row['row']
+        c = row['col']
+        v = row[variable]
+        plate_map.loc[r,c]=v
+    plate_map = plate_map.fillna(0)
+    return pd.DataFrame(plate_map), min_max
+
+def plot_plates(df, variable, grouping, min_max, cmap):
+    try:
+        plates = np.unique(df['plate'], return_counts=False)
+    except:
+        try:
+            df[['plate', 'row', 'col']] = df['prc'].str.split('_', expand=True)
+            df = pd.DataFrame(df)
+            plates = np.unique(df['plate'], return_counts=False)
+        except:
+            next
+    #plates = np.unique(df['plate'], return_counts=False)
+    nr_of_plates = len(plates)
+    print('nr_of_plates:',nr_of_plates)
+    # Calculate the number of rows and columns for the subplot grid
+    if nr_of_plates in [1, 2, 3, 4]:
+        n_rows, n_cols = 1, 4
+    elif nr_of_plates in [5, 6, 7, 8]:
+        n_rows, n_cols = 2, 4
+    elif nr_of_plates in [9, 10, 11, 12]:
+        n_rows, n_cols = 3, 4
+    elif nr_of_plates in [13, 14, 15, 16]:
+        n_rows, n_cols = 4, 4
+
+    # Create the subplot grid with the specified number of rows and columns
+    fig, ax = plt.subplots(n_rows, n_cols, figsize=(40, 5 * n_rows))
+
+    # Flatten the axes array to a one-dimensional array
+    ax = ax.flatten()
+
+    # Loop over each plate and plot the heatmap
+    for index, plate in enumerate(plates):
+        plate_number = plate
+        plate_map, min_max = generate_plate_heatmap(df=df, plate_number=plate_number, variable=variable, grouping=grouping, min_max=min_max)
+        if index == 0:
+            print('plate_number:',plate_number,'minimum:',min_max[0], 'maximum:',min_max[1])
+        # Plot the heatmap on the appropriate subplot
+        sns.heatmap(plate_map, cmap=cmap, vmin=min_max[0], vmax=min_max[1], ax=ax[index])
+        ax[index].set_title(plate_number)
+
+    # Remove any empty subplots
+    for i in range(nr_of_plates, n_rows * n_cols):
+        fig.delaxes(ax[i])
+
+    # Adjust the spacing between the subplots
+    plt.subplots_adjust(wspace=0.1, hspace=0.4)
+
+    # Show the plot
+    plt.show()
+    print()
+    return
+
+def count_mismatches(seq1, seq2, align_length=10):
+    alignments = pairwise2.align.globalxx(seq1, seq2)
+    # choose the first alignment (there might be several with the same score)
+    alignment = alignments[0]
+    # alignment is a tuple (seq1_aligned, seq2_aligned, score, begin, end)
+    seq1_aligned, seq2_aligned, score, begin, end = alignment
+    # Determine the start of alignment (first position where at least align_length bases are the same)
+    start_of_alignment = next(i for i in range(len(seq1_aligned) - align_length + 1) 
+                              if seq1_aligned[i:i+align_length] == seq2_aligned[i:i+align_length])
+    # Trim the sequences to the same length from the start of the alignment
+    seq1_aligned = seq1_aligned[start_of_alignment:]
+    seq2_aligned = seq2_aligned[start_of_alignment:]
+    # Trim the sequences to be of the same length (from the end)
+    min_length = min(len(seq1_aligned), len(seq2_aligned))
+    seq1_aligned = seq1_aligned[:min_length]
+    seq2_aligned = seq2_aligned[:min_length]
+    mismatches = sum(c1 != c2 for c1, c2 in zip(seq1_aligned, seq2_aligned))
+    return mismatches
+    
+
+def get_sequence_data(r1,r2):
+    forward_regex = re.compile(r'^(...GGTGCCACTT)TTTCAAGTTG.*?TTCTAGCTCT(AAAAC[A-Z]{18,22}AACTT)GACATCCCCA.*?AAGGCAAACA(CCCCCTTCGG....).*') 
+    r1fd = forward_regex.search(r1)
+    reverce_regex = re.compile(r'^(...CCGAAGGGGG)TGTTTGCCTT.*?TGGGGATGTC(AAGTT[A-Z]{18,22}GTTTT)AGAGCTAGAA.*?CAACTTGAAA(AAGTGGCACC...).*') 
+    r2fd = reverce_regex.search(r2)
+    rc_r1 = reverse_complement(r1)
+    rc_r2 = reverse_complement(r2) 
+    if all(var is not None for var in [r1fd, r2fd]):
+        try:
+            r1_mis_matches, _ = count_mismatches(seq1=r1, seq2=rc_r2, align_length=5)
+            r2_mis_matches, _ = count_mismatches(seq1=r2, seq2=rc_r1, align_length=5)
+        except:
+            r1_mis_matches = None
+            r2_mis_matches = None
+        column_r1 = reverse_complement(r1fd[1])
+        sgrna_r1 = r1fd[2]
+        platerow_r1 = r1fd[3]
+        column_r2 = r2fd[3]
+        sgrna_r2 = reverse_complement(r2fd[2])
+        platerow_r2 = reverse_complement(r2fd[1])+'N'
+
+        data_dict = {'r1_plate_row':platerow_r1,
+                     'r1_col':column_r1,
+                     'r1_gRNA':sgrna_r1,
+                     'r1_read':r1,
+                     'r2_plate_row':platerow_r2,
+                     'r2_col':column_r2,
+                     'r2_gRNA':sgrna_r2,
+                     'r2_read':r2,
+                     'r1_r2_rc_mismatch':r1_mis_matches,
+                     'r2_r1_rc_mismatch':r2_mis_matches,
+                     'r1_len':len(r1),
+                     'r2_len':len(r2)}
+    else:
+        try:
+            r1_mis_matches, _ = count_mismatches(r1, rc_r2, align_length=5)
+            r2_mis_matches, _ = count_mismatches(r2, rc_r1, align_length=5)
+        except:
+            r1_mis_matches = None
+            r2_mis_matches = None
+        data_dict = {'r1_plate_row':None,
+             'r1_col':None,
+             'r1_gRNA':None,
+             'r1_read':r1,
+             'r2_plate_row':None,
+             'r2_col':None,
+             'r2_gRNA':None,
+             'r2_read':r2,
+             'r1_r2_rc_mismatch':r1_mis_matches,
+             'r2_r1_rc_mismatch':r2_mis_matches,
+             'r1_len':len(r1),
+             'r2_len':len(r2)}
+
+    return data_dict
+
+def get_read_data(identifier, prefix):
+    if identifier.startswith("@"):
+        parts = identifier.split(" ")
+        # The first part contains the instrument, run number, flowcell ID, lane, tile, and coordinates
+        instrument, run_number, flowcell_id, lane, tile, x_pos, y_pos = parts[0][1:].split(":")
+        # The second part contains the read number, filter status, control number, and sample number
+        read, is_filtered, control_number, sample_number = parts[1].split(":")
+        rund_data_dict = {'instrument':instrument, 
+                          'run_number':run_number, 
+                          'flowcell_id':flowcell_id, 
+                          'lane':lane, 
+                          'tile':tile, 
+                          'x_pos':x_pos, 
+                          'y_pos':y_pos, 
+                          'read':read, 
+                          'is_filtered':is_filtered, 
+                          'control_number':control_number, 
+                          'sample_number':sample_number}
+        modified_dict = {prefix + key: value for key, value in rund_data_dict.items()}
+    return modified_dict
+
+def pos_dict(string):
+    pos_dict = {}
+    for i, char in enumerate(string):
+        if char not in pos_dict:
+            pos_dict[char] = [i]
+        else:
+            pos_dict[char].append(i)
+    return pos_dict
+
+def truncate_read(seq,qual,target):
+    index = seq.find(target)
+    end = len(seq)-(3+len(target))
+    if index != -1: # If the sequence is found
+        if index-3 >= 0:
+            seq = seq[index-3:]
+            qual = qual[index-3:]
+
+    return seq, qual
+
+def equalize_lengths(seq1, seq2, pad_char='N'):
+    len_diff = len(seq1) - len(seq2)
+
+    if len_diff > 0:  # seq1 is longer
+        seq2 += pad_char * len_diff  # pad seq2 with 'N's
+    elif len_diff < 0:  # seq2 is longer
+        seq1 += pad_char * (-len_diff)  # pad seq1 with 'N's
+
+    return seq1, seq2
+
+def get_read_data(identifier, prefix):
+    if identifier.startswith("@"):
+        parts = identifier.split(" ")
+        # The first part contains the instrument, run number, flowcell ID, lane, tile, and coordinates
+        instrument, run_number, flowcell_id, lane, tile, x_pos, y_pos = parts[0][1:].split(":")
+        # The second part contains the read number, filter status, control number, and sample number
+        read, is_filtered, control_number, sample_number = parts[1].split(":")
+        rund_data_dict = {'instrument':instrument, 
+                          'x_pos':x_pos, 
+                          'y_pos':y_pos}
+        modified_dict = {prefix + key: value for key, value in rund_data_dict.items()}
+    return modified_dict
+
+def extract_barecodes(r1_fastq, r2_fastq, csv_loc, chunk_size=100000):
+    data_chunk = []
+    # Open both FASTQ files.
+    with open(r1_fastq) as r1_file, open(r2_fastq) as r2_file:
+        index = 0
+        save_index = 0
+        while True:
+            index += 1
+            start = time.time()
+            # Read 4 lines at a time
+            r1_identifier = r1_file.readline().strip()
+            r1_sequence = r1_file.readline().strip()
+            r1_plus = r1_file.readline().strip()
+            r1_quality = r1_file.readline().strip()
+            r2_identifier = r2_file.readline().strip()
+            r2_sequence = r2_file.readline().strip()
+            r2_sequence = reverse_complement(r2_sequence)
+            r2_sequence = r2_sequence
+            r2_plus = r2_file.readline().strip()
+            r2_quality = r2_file.readline().strip()
+            r2_quality = r2_quality
+            if not r1_identifier or not r2_identifier:
+                break
+            #if index > 100:
+            #    break
+            target = 'GGTGCCACTT'
+            r1_sequence, r1_quality = truncate_read(r1_sequence, r1_quality, target)
+            r2_sequence, r2_quality = truncate_read(r2_sequence, r2_quality, target)
+            r1_sequence, r2_sequence = equalize_lengths(r1_sequence, r2_sequence, pad_char='N')
+            r1_quality, r2_quality = equalize_lengths(r1_quality, r2_quality, pad_char='-')
+            alignments = pairwise2.align.globalxx(r1_sequence, r2_sequence)
+            alignment = alignments[0]
+            score = alignment[2]
+            column = None
+            platerow = None
+            grna = None
+            if score >= 125:
+                aligned_r1 = alignment[0]
+                aligned_r2 = alignment[1]
+                position_dict = {i+1: (base1, base2) for i, (base1, base2) in enumerate(zip(aligned_r1, aligned_r2))}
+                phred_quality1 = [ord(char) - 33 for char in r1_quality]
+                phred_quality2 = [ord(char) - 33 for char in r2_quality]
+                r1_q_dict = {i+1: quality for i, quality in enumerate(phred_quality1)}
+                r2_q_dict = {i+1: quality for i, quality in enumerate(phred_quality2)}
+                read = ''
+                for key in sorted(position_dict.keys()):
+                    if position_dict[key][0] != '-' and (position_dict[key][1] == '-' or r1_q_dict.get(key, 0) >= r2_q_dict.get(key, 0)):
+                        read = read + position_dict[key][0]
+                    elif position_dict[key][1] != '-' and (position_dict[key][0] == '-' or r2_q_dict.get(key, 0) > r1_q_dict.get(key, 0)):
+                        read = read + position_dict[key][1]
+                pattern = re.compile(r'^(...GGTGC)CACTT.*GCTCT(TAAAC[A-Z]{18,22}AACTT)GACAT.*CCCCC(TTCGG....).*')
+                regex_patterns = pattern.search(read)
+                if all(var is not None for var in [regex_patterns]):
+                    column = regex_patterns[1]
+                    grna = reverse_complement(regex_patterns[2])
+                    platerow = reverse_complement(regex_patterns[3])
+            elif score < 125:
+                read = r1_sequence
+                pattern = re.compile(r'^(...GGTGC)CACTT.*GCTCT(TAAAC[A-Z]{18,22}AACTT)GACAT.*CCCCC(TTCGG....).*')
+                regex_patterns = pattern.search(read)
+                if all(var is not None for var in [regex_patterns]):
+                    column = regex_patterns[1]
+                    grna = reverse_complement(regex_patterns[2])
+                    platerow = reverse_complement(regex_patterns[3])
+                    #print('2', platerow)
+            data_dict = {'read':read,'column':column,'platerow':platerow,'grna':grna, 'score':score}
+            end = time.time()
+            if data_dict.get('grna') is not None:
+                save_index += 1
+                r1_rund_data_dict = get_read_data(r1_identifier, prefix='r1_')
+                r2_rund_data_dict = get_read_data(r2_identifier, prefix='r2_')
+                r1_rund_data_dict.update(r2_rund_data_dict)
+                r1_rund_data_dict.update(data_dict)
+                r1_rund_data_dict['r1_quality'] = r1_quality
+                r1_rund_data_dict['r2_quality'] = r2_quality
+                data_chunk.append(r1_rund_data_dict)
+                print(f'Processed reads: {index} Found barecodes in {save_index} Time/read: {end - start}', end='\r', flush=True)
+                if save_index % chunk_size == 0:  # Every `chunk_size` reads, write to the CSV
+                    if not os.path.isfile(csv_loc):
+                        df = pd.DataFrame(data_chunk)
+                        df.to_csv(csv_loc, index=False)
+                    else:
+                        df = pd.DataFrame(data_chunk)
+                        df.to_csv(csv_loc, mode='a', header=False, index=False)
+                    data_chunk = []  # Clear the chunk
+                    
+def split_fastq(input_fastq, output_base, num_files):
+    # Create file objects for each output file
+    outputs = [open(f"{output_base}_{i}.fastq", "w") for i in range(num_files)]
+    with open(input_fastq, "r") as f:
+        # Initialize a counter for the lines
+        line_counter = 0
+        for line in f:
+            # Determine the output file
+            output_file = outputs[line_counter // 4 % num_files]
+            # Write the line to the appropriate output file
+            output_file.write(line)
+            # Increment the line counter
+            line_counter += 1
+    # Close output files
+    for output in outputs:
+        output.close()
+
+def process_barecodes(df):
+    print('==== Preprocessing barecodes ====')
+    plate_ls = []
+    row_ls = [] 
+    column_ls = []
+    grna_ls = []
+    read_ls = []
+    score_ls = []
+    match_score_ls = []
+    index_ls = []
+    index = 0
+    print_every = 100
+    for i,row in df.iterrows():
+        index += 1
+        r1_instrument=row['r1_instrument']
+        r1_x_pos=row['r1_x_pos']
+        r1_y_pos=row['r1_y_pos']
+        r2_instrument=row['r2_instrument']
+        r2_x_pos=row['r2_x_pos']
+        r2_y_pos=row['r2_y_pos']
+        read=row['read']
+        column=row['column']
+        platerow=row['platerow']
+        grna=row['grna']
+        score=row['score']
+        r1_quality=row['r1_quality']
+        r2_quality=row['r2_quality']
+        if r1_x_pos == r2_x_pos:
+            if r1_y_pos == r2_y_pos:
+                match_score = 0
+                
+                if grna.startswith('AAGTT'):
+                    match_score += 0.5
+                if column.endswith('GGTGC'):
+                    match_score += 0.5
+                if platerow.endswith('CCGAA'):
+                    match_score += 0.5
+                index_ls.append(index)
+                match_score_ls.append(match_score)
+                score_ls.append(score)
+                read_ls.append(read)
+                plate_ls.append(platerow[:2])
+                row_ls.append(platerow[2:4])
+                column_ls.append(column[:3])
+                grna_ls.append(grna)
+                if index % print_every == 0:
+                    print(f'Processed reads: {index}', end='\r', flush=True)
+    df = pd.DataFrame()
+    df['index'] = index_ls
+    df['score'] = score_ls
+    df['match_score'] = match_score_ls
+    df['plate'] = plate_ls
+    df['row'] = row_ls
+    df['col'] = column_ls
+    df['seq'] = grna_ls
+    df_high_score = df[df['score']>=125]
+    df_low_score = df[df['score']<125]
+    print(f'', flush=True)
+    print(f'Found {len(df_high_score)} high score reads;Found {len(df_low_score)} low score reads')
+    return df, df_high_score, df_low_score
+
+def find_grna(df, grna_df):
+    print('==== Finding gRNAs ====')
+    seqs = list(set(df.seq.tolist()))
+    seq_ls = []
+    grna_ls = []
+    index = 0
+    print_every = 1000
+    for grna in grna_df.Seq.tolist():
+        reverse_regex = re.compile(r'.*({}).*'.format(grna))
+        for seq in seqs:
+            index += 1
+            if index % print_every == 0:
+                print(f'Processed reads: {index}', end='\r', flush=True)
+            found_grna = reverse_regex.search(seq)
+            if found_grna is None:
+                seq_ls.append('error')
+                grna_ls.append('error')
+            else:
+                seq_ls.append(found_grna[0])
+                grna_ls.append(found_grna[1])
+    grna_dict = dict(zip(seq_ls, grna_ls))
+    df = df.assign(grna_seq=df['seq'].map(grna_dict).fillna('error'))
+    print(f'', flush=True)
+    return df
+
+def map_unmapped_grnas(df):
+    print('==== Mapping lost gRNA barecodes ====')
+    def similar(a, b):
+        return SequenceMatcher(None, a, b).ratio()
+    index = 0
+    print_every = 100
+    sequence_list = df[df['grna_seq'] != 'error']['seq'].unique().tolist()
+    grna_error = df[df['grna_seq']=='error']
+    df = grna_error.copy()
+    similarity_dict = {}
+    #change this so that it itterates throug each well
+    for idx, row in df.iterrows():
+        matches = 0
+        match_string = None
+        for string in sequence_list:
+            index += 1
+            if index % print_every == 0:
+                print(f'Processed reads: {index}', end='\r', flush=True)
+            ratio = similar(row['seq'], string)
+            # check if only one character is different
+            if ratio > ((len(row['seq']) - 1) / len(row['seq'])):
+                matches += 1
+                if matches > 1: # if we find more than one match, we break and don't add anything to the dictionary
+                    break
+                match_string = string
+        if matches == 1: # only add to the dictionary if there was exactly one match
+            similarity_dict[row['seq']] = match_string
+    return similarity_dict
+
+def translate_barecodes(df, grna_df, map_unmapped=False):
+    print('==== Translating barecodes ====')
+    if map_unmapped:
+        similarity_dict = map_unmapped_grnas(df)
+        df = df.assign(seq=df['seq'].map(similarity_dict).fillna('error'))
+    df = df.groupby(['plate','row', 'col'])['grna_seq'].value_counts().reset_index(name='count')
+    grna_dict = grna_df.set_index('Seq')['gene'].to_dict()
+    
+    plate_barcodes = {'AA':'p1','TT':'p2','CC':'p3','GG':'p4','AT':'p5','TA':'p6','CG':'p7','GC':'p8'}
+    
+    row_barcodes = {'AA':'r1','AT':'r2','AC':'r3','AG':'r4','TT':'r5','TA':'r6','TC':'r7','TG':'r8',
+                    'CC':'r9','CA':'r10','CT':'r11','CG':'r12','GG':'r13','GA':'r14','GT':'r15','GC':'r16'}
+    
+    col_barcodes = {'AAA':'c1','TTT':'c2','CCC':'c3','GGG':'c4','AAT':'c5','AAC':'c6','AAG':'c7',
+                    'TTA':'c8','TTC':'c9','TTG':'c10','CCA':'c11','CCT':'c12','CCG':'c13','GGA':'c14',
+                    'CCT':'c15','GGC':'c16','ATT':'c17','ACC':'c18','AGG':'c19','TAA':'c20','TCC':'c21',
+                    'TGG':'c22','CAA':'c23','CGG':'c24'}
+
+    
+    df['plate'] = df['plate'].map(plate_barcodes)
+    df['row'] = df['row'].map(row_barcodes)
+    df['col'] = df['col'].map(col_barcodes)
+    df['grna'] = df['grna_seq'].map(grna_dict)
+    df['gene'] = df['grna'].str.split('_').str[1]
+    df = df.fillna('error')
+    df['prc'] = df['plate']+'_'+df['row']+'_'+df['col']
+    df = df[df['count']>=2]
+    error_count = df[df.apply(lambda row: row.astype(str).str.contains('error').any(), axis=1)].shape[0]
+    plate_error = df['plate'].str.contains('error').sum()/len(df)
+    row_error = df['row'].str.contains('error').sum()/len(df)
+    col_error = df['col'].str.contains('error').sum()/len(df)
+    grna_error = df['grna'].str.contains('error').sum()/len(df)
+    print(f'Matched: {len(df)} rows; Errors: plate:{plate_error*100:.3f}% row:{row_error*100:.3f}% column:{col_error*100:.3f}% gRNA:{grna_error*100:.3f}%')
+    return df
+
+def vert_horiz(v, h, n_col):
+    h = h+1
+    if h not in [*range(0,n_col)]:
+        v = v+1
+        h = 0
+    return v,h
+                                            
+def plot_data(df, v, h, color, n_col, ax, x_axis, y_axis, fontsize=12, lw=2, ls='-', log_x=False, log_y=False, title=None):
+    ax[v, h].plot(df[x_axis], df[y_axis], ls=ls, lw=lw, color=color, label=y_axis)
+    ax[v, h].set_title(None)
+    ax[v, h].set_xlabel(None)
+    ax[v, h].set_ylabel(None)
+    ax[v, h].legend(fontsize=fontsize)
+    
+    if log_x:
+        ax[v, h].set_xscale('log')
+    if log_y:
+        ax[v, h].set_yscale('log')
+    v,h =vert_horiz(v, h, n_col)
+    return v, h  
+
+def test_error(df, min_=25,max_=3025, metric='count',log_x=False, log_y=False):
+    max_ = max_+min_
+    step = math.sqrt(min_)
+    plate_error_ls = []
+    col_error_ls = []
+    row_error_ls = []
+    grna_error_ls = []
+    prc_error_ls = []
+    total_error_ls = []
+    temp_len_ls = []
+    val_ls = []
+    df['sum_count'] = df.groupby('prc')['count'].transform('sum')
+    df['fraction'] = df['count'] / df['sum_count']
+    if metric=='fraction':
+        range_ = np.arange(min_, max_, step).tolist()
+    if metric=='count':
+        range_ = [*range(int(min_),int(max_),int(step))]
+    for val in range_:
+        temp = pd.DataFrame(df[df[metric]>val])
+        temp_len = len(temp)
+        if temp_len == 0:
+            break
+        temp_len_ls.append(temp_len)
+        error_count = temp[temp.apply(lambda row: row.astype(str).str.contains('error').any(), axis=1)].shape[0]/len(temp)
+        plate_error = temp['plate'].str.contains('error').sum()/temp_len
+        row_error = temp['row'].str.contains('error').sum()/temp_len
+        col_error = temp['col'].str.contains('error').sum()/temp_len
+        prc_error = temp['prc'].str.contains('error').sum()/temp_len
+        grna_error = temp['gene'].str.contains('error').sum()/temp_len
+        #print(error_count, plate_error, row_error, col_error, prc_error, grna_error)
+        val_ls.append(val)
+        total_error_ls.append(error_count)
+        plate_error_ls.append(plate_error)
+        row_error_ls.append(row_error)
+        col_error_ls.append(col_error)
+        prc_error_ls.append(prc_error)
+        grna_error_ls.append(grna_error)
+    df2 = pd.DataFrame()
+    df2['val'] = val_ls
+    df2['plate'] = plate_error_ls
+    df2['row'] = row_error_ls
+    df2['col'] = col_error_ls
+    df2['gRNA'] = grna_error_ls
+    df2['prc'] = prc_error_ls
+    df2['total'] = total_error_ls
+    df2['len'] = temp_len_ls
+                                 
+    n_row, n_col = 2, 7
+    v, h, lw, ls, color = 0, 0, 1, '-', 'teal'
+    fig, ax = plt.subplots(n_row, n_col, figsize=(n_col*5, n_row*5))
+    
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='total',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='prc',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='plate',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='row',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='col',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='gRNA',log_x=log_x, log_y=log_y)
+    v, h = plot_data(df=df2, v=v, h=h, color=color, n_col=n_col, ax=ax, x_axis='val', y_axis='len',log_x=log_x, log_y=log_y)
+    
+def generate_fraction_map(df, gene_column, min_=10, plates=['p1','p2','p3','p4'], metric = 'count', plot=False):
+    df['prcs'] = df['prc']+''+df['grna_seq']
+    df['gene'] = df['grna'].str.split('_').str[1]
+    if metric == 'count':
+        df = pd.DataFrame(df[df['count']>min_])
+    df = df[~(df == 'error').any(axis=1)]
+    df = df[df['plate'].isin(plates)]
+    gRNA_well_count = df.groupby('prc')['prcs'].transform('nunique')
+    df['gRNA_well_count'] = gRNA_well_count
+    df = df[df['gRNA_well_count']>=2]
+    df = df[df['gRNA_well_count']<=100]
+    well_sum = df.groupby('prc')['count'].transform('sum')
+    df['well_sum'] = well_sum
+    df['gRNA_fraction'] = df['count']/df['well_sum']
+    if metric == 'fraction':
+        df = pd.DataFrame(df[df['gRNA_fraction']>=min_])
+        df = df[df['plate'].isin(plates)]
+        gRNA_well_count = df.groupby('prc')['prcs'].transform('nunique')
+        df['gRNA_well_count'] = gRNA_well_count
+        well_sum = df.groupby('prc')['count'].transform('sum')
+        df['well_sum'] = well_sum
+        df['gRNA_fraction'] = df['count']/df['well_sum']
+    if plot:
+        print('gRNAs/well')
+        plot_plates(df=df, variable='gRNA_well_count', grouping='mean', min_max='allq', cmap='viridis')
+        print('well read sum')
+        plot_plates(df=df, variable='well_sum', grouping='mean', min_max='allq', cmap='viridis')
+    genes = df[gene_column].unique().tolist()
+    wells = df['prc'].unique().tolist()
+    print('numer of genes:',len(genes),'numer of wells:', len(wells))
+    independent_variables = pd.DataFrame(columns=genes, index = wells)
+    for index, row in df.iterrows():
+        prc = row['prc']
+        gene = row[gene_column]
+        fraction = row['gRNA_fraction']
+        independent_variables.loc[prc,gene]=fraction
+    independent_variables = independent_variables.fillna(0.0)
+    independent_variables['sum'] = independent_variables.sum(axis=1)
+    independent_variables = independent_variables[independent_variables['sum']==1.0]
+    independent_variables = independent_variables.drop('sum', axis=1)
+    independent_variables.index.name = 'prc'
+    independent_variables = independent_variables.loc[:, (independent_variables.sum() != 0)]
+    return independent_variables
+
+# Check if filename or path
+def split_filenames(df, filename_column):
+    plate_ls = []
+    well_ls = []
+    col_ls = []
+    row_ls = []
+    field_ls = []
+    obj_ls = []
+    ls = df[filename_column].tolist()
+    if '/' in ls[0]:
+        file_list = [os.path.basename(path) for path in ls] 
+    else:
+        file_list = ls
+    print('first file',file_list[0])
+    for filename in file_list:
+        plate = filename.split('_')[0]
+        plate = plate.split('plate')[1]
+        well = filename.split('_')[1]
+        field = filename.split('_')[2]
+        object_nr = filename.split('_')[3]
+        object_nr = object_nr.split('.')[0]
+        object_nr = 'o'+str(object_nr)
+        if re.match('A..', well):
+            row = 'r1'
+        if re.match('B..', well):
+            row = 'r2'
+        if re.match('C..', well):
+            row = 'r3'
+        if re.match('D..', well):
+            row = 'r4'
+        if re.match('E..', well):
+            row = 'r5'
+        if re.match('F..', well):
+            row = 'r6'
+        if re.match('G..', well):
+            row = 'r7'
+        if re.match('H..', well):
+            row = 'r8'
+        if re.match('I..', well):
+            row = 'r9'
+        if re.match('J..', well):
+            row = 'r10'
+        if re.match('K..', well):
+            row = 'r11'
+        if re.match('L..', well):
+            row = 'r12'
+        if re.match('M..', well):
+            row = 'r13'
+        if re.match('N..', well):
+            row = 'r14'
+        if re.match('O..', well):
+            row = 'r15'
+        if re.match('P..', well):
+            row = 'r16'
+        if re.match('.01', well):
+            col = 'c1'
+        if re.match('.02', well):
+            col = 'c2'
+        if re.match('.03', well):
+            col = 'c3'
+        if re.match('.04', well):
+            col = 'c4'
+        if re.match('.05', well):
+            col = 'c5'
+        if re.match('.06', well):
+            col = 'c6'
+        if re.match('.07', well):
+            col = 'c7'
+        if re.match('.08', well):
+            col = 'c8'
+        if re.match('.09', well):
+            col = 'c9'
+        if re.match('.10', well):
+            col = 'c10'
+        if re.match('.11', well):
+            col = 'c11'
+        if re.match('.12', well):
+            col = 'c12'
+        if re.match('.13', well):
+            col = 'c13'
+        if re.match('.14', well):
+            col = 'c14'
+        if re.match('.15', well):
+            col = 'c15'
+        if re.match('.16', well):
+            col = 'c16'
+        if re.match('.17', well):
+            col = 'c17'
+        if re.match('.18', well):
+            col = 'c18'
+        if re.match('.19', well):
+            col = 'c19'
+        if re.match('.20', well):
+            col = 'c20'
+        if re.match('.21', well):
+            col = 'c21'
+        if re.match('.22', well):
+            col = 'c22'
+        if re.match('.23', well):
+            col = 'c23'
+        if re.match('.24', well):
+            col = 'c24'
+        plate_ls.append(plate)
+        well_ls.append(well)
+        field_ls.append(field)
+        obj_ls.append(object_nr)
+        row_ls.append(row)
+        col_ls.append(col)
+    df['file'] = ls
+    df['plate'] = plate_ls
+    df['well'] = well_ls
+    df['row'] = row_ls
+    df['col'] = col_ls
+    df['field'] = field_ls
+    df['obj'] = obj_ls
+    df['plate_well'] = df['plate']+'_'+df['well']
+    df = df.set_index(filename_column)
+    return df
+
+def rename_plate_metadata(df):
+    try:
+        df = df.drop(['plateID'], axis=1)
+        df = df.drop(['rowID'], axis=1)
+        df = df.drop(['columnID'], axis=1)
+        df = df.drop(['plate_row_col'], axis=1)
+        df = df.drop(['Unnamed: 0'], axis=1)
+        df = df.drop(['Unnamed: 0.1'], axis=1)
+    except:
+        next
+    
+    df['plate'] = df['plate'].astype('string')
+    df.plate.replace('1', 'A', inplace=True)
+    df.plate.replace('2', 'B', inplace=True)
+    df.plate.replace('3', 'C', inplace=True)
+    df.plate.replace('4', 'D', inplace=True)
+    df.plate.replace('5', 'E', inplace=True)
+    df.plate.replace('6', 'F', inplace=True)
+    df.plate.replace('7', 'G', inplace=True)
+    df.plate.replace('8', 'H', inplace=True)
+    df.plate.replace('9', 'I', inplace=True)
+    df.plate.replace('10', 'J', inplace=True)
+
+    df.plate.replace('A', 'p1', inplace=True)# 1 - 1
+    df.plate.replace('B', 'p2', inplace=True)# 2 - 2
+    df.plate.replace('C', 'p3', inplace=True)# 3 - 3
+    df.plate.replace('E', 'p4', inplace=True)# 5 - 4
+    
+    df.plate.replace('F', 'p5', inplace=True)# 6 - 5
+    df.plate.replace('G', 'p6', inplace=True)# 7 - 6
+    df.plate.replace('H', 'p7', inplace=True)# 8 - 7
+    df.plate.replace('I', 'p8', inplace=True)# 9 - 8
+    
+    df['plateID'] = df['plate']
+    
+    df.loc[(df['plateID'].isin(['D'])) & (df['col'].isin(['c1', 'c2', 'c3'])), 'plate'] = 'p1'
+    df.loc[(df['plateID'].isin(['D'])) & (df['col'].isin(['c4', 'c5', 'c6'])), 'plate'] = 'p2'
+    df.loc[(df['plateID'].isin(['D'])) & (df['col'].isin(['c7', 'c8', 'c9'])), 'plate'] = 'p3'
+    df.loc[(df['plateID'].isin(['D'])) & (df['col'].isin(['c10', 'c11', 'c12'])), 'plate'] = 'p4'
+    
+    df.loc[(df['plateID'].isin(['J'])) & (df['col'].isin(['c1', 'c2', 'c3'])), 'plate'] = 'p5'
+    df.loc[(df['plateID'].isin(['J'])) & (df['col'].isin(['c4', 'c5', 'c6'])), 'plate'] = 'p6'
+    df.loc[(df['plateID'].isin(['J'])) & (df['col'].isin(['c7', 'c8', 'c9'])), 'plate'] = 'p7'
+    df.loc[(df['plateID'].isin(['J'])) & (df['col'].isin(['c10', 'c11', 'c12'])), 'plate'] = 'p8'
+    
+    df.loc[(df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c1', 'c4', 'c7', 'c10'])), 'col'] = 'c1'
+    df.loc[(df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c2', 'c5', 'c8', 'c11'])), 'col'] = 'c2'
+    df.loc[(df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c3', 'c6', 'c9', 'c12'])), 'col'] = 'c3'
+    
+    df.loc[(~df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c1'])), 'col'] = 'c25'
+    df.loc[(~df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c2'])), 'col'] = 'c26'
+    df.loc[(~df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c3'])), 'col'] = 'c27'
+    
+    df.loc[(~df['plateID'].isin(['D', 'J'])) & (df['col'].isin(['c1'])), 'col'] = 'c25'
+
+    df = df.drop(['plateID'], axis=1)
+    
+    df = df.loc[~df['plate'].isin(['D', 'J'])]
+
+    screen_cols = ['c1','c2','c3','c4','c5','c6','c7','c8','c9','c10','c11','c12','c13','c14','c15','c16','c17','c18','c19','c20','c21','c22','c23','c24']
+    screen_plates = ['p1','p2','p3','p4']
+    positive_control_plates = ['p5','p6','p7','p8']
+    positive_control_cols = screen_cols
+    negative_control_cols = ['c25','c26','c27']
+    #extra_plates = ['p9','p10']
+    cond_ls = []
+
+    cols = df.col.tolist()
+    for index, plate in enumerate(df.plate.tolist()):
+        co = cols[index]
+        if plate in screen_plates:
+            if co in screen_cols:
+                cond = 'SCREEN'
+            if co in negative_control_cols:
+                cond = 'NC'
+        if plate in positive_control_plates:
+            if co in positive_control_cols:
+                cond = 'PC'
+            if co in negative_control_cols:
+                cond = 'NC'
+        cond_ls.append(cond)
+        
+    df['cond'] = cond_ls
+    df['plate'] = df['plate'].astype('string')
+    df['row'] = df['row'].astype('string')
+    df['col'] = df['col'].astype('string')
+    df['obj'] = df['obj'].astype('string')
+    df['prco'] = df['plate']+'_'+df['row']+'_'+df['col']+'_'+df['field']+'_'+df['obj']
+    df['prc'] = df['plate']+'_'+df['row']+'_'+df['col']
+    df = df.set_index(['prco'], drop=True)
+    df = df.sort_values(by = ['plate'], ascending = [True], na_position = 'first')
+    values, counts = np.unique(df['plate'], return_counts=True)
+    print('plates:', values)
+    print('well count:', counts)
+    return df
+
+def plot_reg_res(df, coef_col='coef', col_p='P>|t|'):
+    df['gene'] = df.index
+    df[coef_col] = pd.to_numeric(df[coef_col], errors='coerce')
+    df[col_p] = pd.to_numeric(df[col_p], errors='coerce')
+    df = df.sort_values(by = [coef_col], ascending = [False], na_position = 'first')
+    df['color'] = 'None'
+    df.loc[df['gene'].str.contains('239740'), 'color'] = '239740'
+    df.loc[df['gene'].str.contains('205250'), 'color'] = '205250'
+    
+    df.loc[df['gene'].str.contains('000000'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000001'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000002'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000003'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000004'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000005'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000006'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000007'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000008'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000009'), 'color'] = '000000'
+    df.loc[df['gene'].str.contains('000010'), 'color'] = '000000'
+    fig, ax = plt.subplots(figsize=(10,10))
+    df.loc[df[col_p] == 0.000, col_p] = 0.001
+    df['logp'] = -np.log10(df[col_p])
+    sns.scatterplot(data = df, x = coef_col, y = 'logp', legend = False, ax = ax,
+                    hue= 'color', hue_order = ['239740','205250','None', '000000'],
+                    palette = ['purple', 'teal', 'lightgrey', 'black'],
+                    size = 'color', sizes = (100, 10))
+    g14 = df[df['gene'].str.contains('239740')]
+    r18 = df[df['gene'].str.contains('205250')]
+    res = pd.concat([g14, r18], axis=0)
+    res = res[[coef_col, col_p]]
+    print(res)
+    return df, res
+
+def reg_model(iv_loc,dv_loc):
+    independent_variables = pd.read_csv(iv_loc)
+    dependent_variable = pd.read_csv(dv_loc)
+    independent_variables = independent_variables.set_index('prc')
+    columns = independent_variables.columns
+    new_columns = [col.replace('TGGT1_', '') for col in columns]
+    independent_variables.columns = new_columns
+
+    dependent_variable = dependent_variable.set_index('prc')
+
+    reg_input = pd.DataFrame(pd.merge(independent_variables, dependent_variable, left_index=True, right_index=True))
+    reg_input = reg_input.dropna(axis=0, how='any')
+    reg_input = reg_input.dropna(axis=1, how='any')
+    print('Number of wells',len(reg_input))
+    x = reg_input.drop(['score'], axis=1)
+    x = sm.add_constant(x)
+    y = np.log10(reg_input['score']+1)
+    model = sm.OLS(y, x).fit()
+    predictions = model.predict(x)
+    results_summary = model.summary()
+    print(results_summary)
+    results_as_html = results_summary.tables[1].as_html()
+    results_df = pd.read_html(results_as_html, header=0, index_col=0)[0]
+    df, res = plot_reg_res(df=results_df)
+    return df, res
+
+def mixed_model(iv_loc,dv_loc):
+    independent_variables = pd.read_csv(iv_loc)
+    dependent_variable = pd.read_csv(dv_loc)
+    independent_variables = independent_variables.set_index('prc')
+    columns = independent_variables.columns
+    new_columns = [col.replace('TGGT1_', '') for col in columns]
+    independent_variables.columns = new_columns
+    dependent_variable = dependent_variable.set_index('prc')
+    reg_input = pd.DataFrame(pd.merge(independent_variables, dependent_variable, left_index=True, right_index=True))
+    reg_input = reg_input.dropna(axis=0, how='any')
+
+    y = np.log10(reg_input['score']+1)
+    X = reg_input.drop('score', axis=1)
+    X.columns = pd.MultiIndex.from_tuples([tuple(col.split('_')) for col in X.columns],
+                                          names=['main_variable', 'sub_variable'])
+    # Melt the DataFrame
+    X_long = X.melt(ignore_index=False, var_name=['main_variable', 'sub_variable'], value_name='value')
+    X_long = X_long[X_long['value']>0]
+
+    # Create a new column to represent the nested structure of gRNA within gene
+    X_long['gene_gRNA'] = X_long['main_variable'].astype(str) + "_" + X_long['sub_variable'].astype(str)
+
+    # Add 'score' to the DataFrame
+    X_long['score'] = y
+
+    # Create and convert the plate, row, and column variables to type category
+    X_long.reset_index(inplace=True)  
+    split_values = X_long['prc'].str.split('_', expand=True)
+    X_long[['plate', 'row', 'col']] = split_values
+    X_long['plate'] = X_long['plate'].str[1:]
+    X_long['plate'] = X_long['plate'].astype(int)
+    X_long['row'] = X_long['row'].str[1:]
+    X_long['row'] = X_long['row'].astype(int)
+    X_long['col'] = X_long['col'].str[1:]
+    X_long['col'] = X_long['col'].astype(int)
+    X_long = X_long.set_index('prc')
+    # Create a new column to represent the nested structure of plate, row, and column
+    X_long['plate_row_col'] = X_long['plate'].astype(str) + "_" + X_long['row'].astype(str) + "_" + X_long['col'].astype(str)
+    n_group = pd.DataFrame(X_long.groupby(['gene_gRNA']).count()['main_variable'])
+    n_group = n_group.rename({'main_variable': 'n_group'}, axis=1)
+    n_group = n_group.reset_index(drop=False)
+    X_long = pd.merge(X_long, n_group, on='gene_gRNA')
+    X_long = X_long[X_long['n_group']>1]
+    #print(X_long.isna().sum())
+    
+    X_long['main_variable'] = X_long['main_variable'].astype('category')
+    X_long['sub_variable'] = X_long['sub_variable'].astype('category')
+    X_long['plate'] = X_long['plate'].astype('category')
+    X_long['row'] = X_long['row'].astype('category')
+    X_long['col'] = X_long['col'].astype('category')
+    X_long = pd.DataFrame(X_long)
+    print(X_long)
+    
+    md = smf.mixedlm("score ~ C(main_variable)", X_long, 
+                 groups=X_long["sub_variable"])
+    
+    # Define your nonlinear function here
+    def nonlinear_function(x, *params):
+        pass  # Implement non linear function here
+
+    mdf = md.fit(method='bfgs', maxiter=1000)
+    print(mdf.summary())
+    summary = mdf.summary()
+    df = pd.DataFrame(summary.tables[1])
+    df, res = plot_reg_res(df, coef_col='Coef.', col_p='P>|z|')
+    return df, res
+
+def calculate_accuracy(df):
+    df.loc[df['pc_score'] <= 0.5, 'pred'] = 0
+    df.loc[df['pc_score'] >= 0.5, 'pred'] = 1
+    df.loc[df['cond'] == 'NC', 'lab'] = 0
+    df.loc[df['cond'] == 'PC', 'lab'] = 1
+    df = df[df['cond'] != 'SCREEN']
+    df_nc = df[df['cond'] != 'NC']
+    df_pc = df[df['cond'] != 'PC']
+    correct = []
+    all_ls = []
+    pred_list = df['pred'].tolist()
+    lab_list = df['lab'].tolist()
+    for i,v in enumerate(pred_list):
+        all_ls.append(1)
+        if v == lab_list[i]:
+            correct.append(1)
+    print('total accuracy',len(correct)/len(all_ls))
+    correct = []
+    all_ls = []
+    pred_list = df_pc['pred'].tolist()
+    lab_list = df_pc['lab'].tolist()
+    for i,v in enumerate(pred_list):
+        all_ls.append(1)
+        if v == lab_list[i]:
+            correct.append(1)
+    print('positives accuracy', len(correct)/len(all_ls))
+    correct = []
+    all_ls = []
+    pred_list = df_nc['pred'].tolist()
+    lab_list = df_nc['lab'].tolist()
+    for i,v in enumerate(pred_list):
+        all_ls.append(1)
+        if v == lab_list[i]:
+            correct.append(1)
+    print('negatives accuracy',len(correct)/len(all_ls))
+
+def preprocess_image_data(df, resnet_loc, min_count=25, metric='mean', plot=True, score='pc_score'):
+    print('number of cells', len(df))
+    resnet_preds = pd.read_csv(resnet_loc)
+    res_df = split_filenames(df=resnet_preds, filename_column='path')
+    pred_df = rename_plate_metadata(df=res_df)
+    pred_df['prcfo'] = pred_df['plate']+'_'+pred_df['row']+'_'+pred_df['col']+'_'+pred_df['field']+'_'+pred_df['obj']
+    print('number of resnet scores', len(df))
+    merged_df = pd.merge(df, pred_df, on='prcfo', how='inner', suffixes=('', '_right'))
+    merged_df = merged_df.rename(columns={'pred': 'pc_score'})
+    
+    merged_df = merged_df[(merged_df['pc_score'] <= 0.25) | (merged_df['pc_score'] >= 0.75)]
+    
+    merged_df['recruitment'] = merged_df['Toxo_channel_1_quartiles75']/merged_df['Cytosol_channel_1_quartiles75']
+    merged_df = pd.DataFrame(merged_df[merged_df['duplicates'] == 1.0])
+    columns_to_drop = [col for col in merged_df.columns if col.endswith('_right')]
+    merged_df = merged_df.drop(columns_to_drop, axis=1)
+    well_group = pd.DataFrame(merged_df.groupby(['prc']).count()['cond'])
+    well_group = well_group.rename({'cond': 'cell_count'}, axis=1)
+    merged_df = pd.merge(merged_df, well_group, on='prc', how='inner', suffixes=('', '_right'))
+    columns_to_drop = [col for col in merged_df.columns if col.endswith('_right')]
+    merged_df = merged_df.drop(columns_to_drop, axis=1)
+    #merged_df = merged_df.drop(['duplicates', 'outlier', 'prcfo.1'], axis=1)
+    merged_df = merged_df.drop(['duplicates', 'prcfo.1'], axis=1)
+    merged_df = pd.DataFrame(merged_df[merged_df['cell_count'] >= min_count])
+    
+    if metric == 'mean':
+        well_scores_score = pd.DataFrame(merged_df.groupby(['prc']).mean()['pc_score'])
+        well_scores_score = well_scores_score.rename({'pc_score': 'mean_pc_score'}, axis=1)
+        well_scores_rec = pd.DataFrame(merged_df.groupby(['prc']).mean()['recruitment'])
+        well_scores_rec = well_scores_rec.rename({'recruitment': 'mean_recruitment'}, axis=1)
+    if metric == 'geomean':
+        well_scores_score = pd.DataFrame(merged_df.groupby(['prc'])['pc_score'].apply(gmean))
+        well_scores_score = well_scores_score.rename({'pc_score': 'mean_pc_score'}, axis=1)
+        well_scores_rec = pd.DataFrame(merged_df.groupby(['prc'])['recruitment'].apply(gmean))
+        well_scores_rec = well_scores_rec.rename({'recruitment': 'mean_recruitment'}, axis=1)
+    if metric == 'median':
+        well_scores_score = pd.DataFrame(merged_df.groupby(['prc']).median()['pc_score'])
+        well_scores_score = well_scores_score.rename({'pc_score': 'mean_pc_score'}, axis=1)
+        well_scores_rec = pd.DataFrame(merged_df.groupby(['prc']).median()['recruitment'])
+        well_scores_rec = well_scores_rec.rename({'recruitment': 'mean_recruitment'}, axis=1)
+    if metric == 'quntile':
+        well_scores_score = pd.DataFrame(merged_df.groupby(['prc']).quantile(0.75)['pc_score'])
+        well_scores_score = well_scores_score.rename({'pc_score': 'mean_pc_score'}, axis=1)
+        well_scores_rec = pd.DataFrame(merged_df.groupby(['prc']).quantile(0.75)['recruitment'])
+        well_scores_rec = well_scores_rec.rename({'recruitment': 'mean_recruitment'}, axis=1)
+    well = pd.DataFrame(pd.DataFrame(merged_df.select_dtypes(include=['object'])).groupby(['prc']).first())
+    well['mean_pc_score'] = well_scores_score['mean_pc_score']
+    well['mean_recruitment'] = well_scores_rec['mean_recruitment']
+    nc = well[well['cond'] == 'NC']
+    max_nc = nc['mean_recruitment'].max()
+    pc = well[well['cond'] == 'PC']
+    screen = well[well['cond'] == 'SCREEN']
+    screen = screen[screen['mean_recruitment'] <= max_nc]
+    if plot:
+        x_axis = 'mean_pc_score'
+        fig, ax = plt.subplots(1,3,figsize=(30,10))
+        sns.histplot(data=nc, x=x_axis, kde=False, stat='density', element="step", ax=ax[0], color='lightgray', log_scale=False)
+        sns.histplot(data=pc, x=x_axis, kde=False, stat='density', element="step", ax=ax[0], color='teal', log_scale=False)
+        sns.histplot(data=screen, x=x_axis, kde=False, stat='density', element="step", ax=ax[1], color='purple', log_scale=False)
+        sns.histplot(data=nc, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='lightgray', log_scale=False)
+        sns.histplot(data=pc, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='teal', log_scale=False)
+        sns.histplot(data=screen, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='purple', log_scale=False)
+        ax[0].set_title('NC vs PC wells')
+        ax[1].set_title('Screen wells')
+        ax[2].set_title('NC vs PC vs Screen wells')
+        ax[0].spines['top'].set_visible(False)
+        ax[0].spines['right'].set_visible(False)
+        ax[1].spines['top'].set_visible(False)
+        ax[1].spines['right'].set_visible(False)
+        ax[2].spines['top'].set_visible(False)
+        ax[2].spines['right'].set_visible(False)
+        ax[0].set_xlim([0, 1])
+        ax[1].set_xlim([0, 1])
+        ax[2].set_xlim([0, 1])
+        loc = '/media/olafsson/umich/matt_graphs/resnet_score_well_av.pdf'
+        fig.savefig(loc, dpi = 600, format='pdf', bbox_inches='tight')
+        x_axis = 'mean_recruitment'
+        fig, ax = plt.subplots(1,3,figsize=(30,10))
+        sns.histplot(data=nc, x=x_axis, kde=False, stat='density', element="step", ax=ax[0], color='lightgray', log_scale=False)
+        sns.histplot(data=pc, x=x_axis, kde=False, stat='density', element="step", ax=ax[0], color='teal', log_scale=False)
+        sns.histplot(data=screen, x=x_axis, kde=False, stat='density', element="step", ax=ax[1], color='purple', log_scale=False)
+        sns.histplot(data=nc, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='lightgray', log_scale=False)
+        sns.histplot(data=pc, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='teal', log_scale=False)
+        sns.histplot(data=screen, x=x_axis, kde=False, stat='density', element="step", ax=ax[2], color='purple', log_scale=False)
+        ax[0].set_title('NC vs PC wells')
+        ax[1].set_title('Screen wells')
+        ax[2].set_title('NC vs PC vs Screen wells')
+        ax[0].spines['top'].set_visible(False)
+        ax[0].spines['right'].set_visible(False)
+        ax[1].spines['top'].set_visible(False)
+        ax[1].spines['right'].set_visible(False)
+        ax[2].spines['top'].set_visible(False)
+        ax[2].spines['right'].set_visible(False)
+        loc = '/media/olafsson/umich/matt_graphs/mean_recruitment_well_av.pdf'
+        fig.savefig(loc, dpi = 600, format='pdf', bbox_inches='tight')
+    plates = ['p1','p2','p3','p4']
+    screen = screen[screen['plate'].isin(plates)]
+    if score == 'pc_score':
+        dv = pd.DataFrame(screen['mean_pc_score'])
+        dv = dv.rename({'mean_pc_score': 'score'}, axis=1)
+    if score == 'recruitment':
+        dv = pd.DataFrame(screen['mean_recruitment'])
+        dv = dv.rename({'mean_recruitment': 'score'}, axis=1)
+    print('dependant variable well count:', len(well))
+    dv_loc = '/media/olafsson/Data2/methods_paper/data/dv.csv'
+    dv.to_csv(dv_loc)
+    calculate_accuracy(df=merged_df)
+    return merged_df, well