app.py

import streamlit as st
import pandas as pd
import primer3
from Bio import SeqIO
import os
from io import StringIO
import matplotlib.pyplot as plt

# Ensure the 'temp' directory exists for saving temporary files
temp_dir = "temp"
os.makedirs(temp_dir, exist_ok=True)

# Streamlit UI setup
st.set_page_config(page_title="PCR Primer Design", page_icon="🧬", layout="wide")
uploaded_file = st.file_uploader("Upload a GenBank file", type=['gb', 'gbk'])

def extract_features_from_genbank(genbank_content, feature_types=['CDS', 'tRNA', 'gene']):
    """Extracts specified features from GenBank content."""
    text_stream = StringIO(genbank_content.decode("utf-8")) if isinstance(genbank_content, bytes) else genbank_content
    record = SeqIO.read(text_stream, "genbank")
    features = {ftype: [] for ftype in feature_types}
    for feature in record.features:
        if feature.type in feature_types:
            features[feature.type].append(feature)
    return features, record

def design_primers_for_region(sequence, product_size_range, num_to_return=5):
    """Design primers for a specific sequence."""
    # Parse the product size range
    size_min, size_max = map(int, product_size_range.split('-'))
    return primer3.bindings.designPrimers(
        {
            'SEQUENCE_TEMPLATE': str(sequence),
            'PRIMER_PRODUCT_SIZE_RANGE': [[size_min, size_max]]
        },
        {
            'PRIMER_OPT_SIZE': 20,
            'PRIMER_MIN_SIZE': 18,
            'PRIMER_MAX_SIZE': 23,
            'PRIMER_OPT_TM': 60.0,
            'PRIMER_MIN_TM': 57.0,
            'PRIMER_MAX_TM': 63.0,
            'PRIMER_MIN_GC': 20.0,
            'PRIMER_MAX_GC': 80.0,
            'PRIMER_NUM_RETURN': num_to_return,
        }
    )

def plot_pcr_product(sequence, primers, feature_length, num_pairs=5):
    """Visualize the PCR product based on primer locations relative to a feature."""
    import matplotlib.pyplot as plt  # Ensure matplotlib is imported here if not already done
    
    # Start the figure for plotting
    plt.figure(figsize=(10, 3))
    plt.plot([0, feature_length], [1, 1], 'b-', label='Selected Feature')  # Blue line represents the selected feature
    
    for i in range(num_pairs):
        left_key = f'PRIMER_LEFT_{i}_POSITION'  # Correct key for left primer position
        right_key = f'PRIMER_RIGHT_{i}_POSITION'  # Correct key for right primer position
        
        # Check if keys exist before trying to access them
        if left_key in primers and right_key in primers:
            left_pos = primers[left_key]
            right_pos = primers[right_key]
            # Draw lines for primers; left primer above (y=1.2) and right primer below (y=0.8) the feature line
            plt.plot([left_pos, left_pos], [1.1, 1.3], 'r-', label='Left Primer' if i == 0 else "")  # Red line represents left primer
            plt.plot([right_pos, right_pos], [0.7, 0.9], 'g-', label='Right Primer' if i == 0 else "")  # Green line represents right primer
            plt.text(left_pos, 1.4, f'L{i+1}', ha='center')  # Label for left primer
            plt.text(right_pos, 0.6, f'R{i+1}', ha='center')  # Label for right primer
    
    plt.ylim(0, 2)  # Set the limits of Y-axis to make the plot clearer
    plt.title('Feature and Primer Positions')
    plt.xlabel('Nucleotide position')
    plt.legend()
    plt.grid(True)
    
    # Use st.pyplot() to display the plot in Streamlit
    st.pyplot(plt)


if uploaded_file is not None:
    genbank_content = StringIO(uploaded_file.getvalue().decode("utf-8"))
    features, record = extract_features_from_genbank(genbank_content)
    feature_type = st.selectbox('Select feature type:', ['CDS', 'tRNA', 'gene'])

    feature_options = [f"{feature.qualifiers.get('gene', [''])[0]} ({feature.location})" for feature in features[feature_type]]
    selected_index = st.selectbox(f'Select a {feature_type}:', options=range(len(feature_options)), format_func=lambda x: feature_options[x])
    selected_feature = features[feature_type][selected_index]

    feature_sequence = selected_feature.extract(record.seq)
    st.write(f"Selected {feature_type} sequence (length: {len(feature_sequence)} bp):")
    st.text(str(feature_sequence))

    product_size_range = st.text_input("Enter desired PCR product size range (e.g., 150-500):", value="150-500")

    if st.button(f'Design Primers for selected {feature_type}'):
        primers = design_primers_for_region(feature_sequence, product_size_range, num_to_return=5)
        
        primer_data = []
        for i in range(5):  # Collect data for 5 primer pairs
            primer_info = {
                'Primer Pair': i + 1,
                'Left Sequence': primers.get(f'PRIMER_LEFT_{i}_SEQUENCE', 'N/A'),
                'Right Sequence': primers.get(f'PRIMER_RIGHT_{i}_SEQUENCE', 'N/A'),
                'Left TM (°C)': primers.get(f'PRIMER_LEFT_{i}_TM', 'N/A'),
                'Right TM (°C)': primers.get(f'PRIMER_RIGHT_{i}_TM', 'N/A'),
                'Left Length': primers.get(f'PRIMER_LEFT_{i}_SIZE', 'N/A'),
                'Right Length': primers.get(f'PRIMER_RIGHT_{i}_SIZE', 'N/A'),
                'PCR Product Size (bp)': primers.get(f'PRIMER_PAIR_{i}_PRODUCT_SIZE', 'N/A')
            }
            if primer_info['Left Sequence'] != 'N/A' and primer_info['Right Sequence'] != 'N/A':
                primer_data.append(primer_info)

        if primer_data:
            primer_df = pd.DataFrame(primer_data)
            st.write('### Designed Primers')
            st.dataframe(primer_df)
            csv = primer_df.to_csv(index=False).encode('utf-8')
            st.download_button(
                "Download Primers as CSV",
                csv,
                "primers.csv",
                "text/csv",
                key='download-csv'
            )
            # Plotting PCR products
            st.write("### Visualization of PCR Products")
            feature_length = len(feature_sequence)  # Length of the selected feature
            plot_pcr_product(feature_sequence, primers, feature_length, num_pairs=5)
        else:
            st.error('No primers were found. Please adjust your parameters and try again.')