import requests import tensorflow as tf import pandas as pd import numpy as np from operator import add from functools import reduce from Bio import SeqIO from Bio.SeqRecord import SeqRecord from Bio.SeqFeature import SeqFeature, FeatureLocation from Bio.Seq import Seq from keras.models import load_model import random # configure GPUs for gpu in tf.config.list_physical_devices('GPU'): tf.config.experimental.set_memory_growth(gpu, enable=True) if len(tf.config.list_physical_devices('GPU')) > 0: tf.config.experimental.set_visible_devices(tf.config.list_physical_devices('GPU')[0], 'GPU') ntmap = {'A': (1, 0, 0, 0), 'C': (0, 1, 0, 0), 'G': (0, 0, 1, 0), 'T': (0, 0, 0, 1) } def get_seqcode(seq): return np.array(reduce(add, map(lambda c: ntmap[c], seq.upper()))).reshape( (1, len(seq), -1)) from keras.models import load_model class DCModelOntar: def __init__(self, ontar_model_dir, is_reg=False): self.model = load_model(ontar_model_dir) def ontar_predict(self, x, channel_first=True): if channel_first: x = x.transpose([0, 2, 3, 1]) yp = self.model.predict(x) return yp.ravel() # Function to predict on-target efficiency and format output def format_prediction_output(targets, model_path): dcModel = DCModelOntar(model_path) formatted_data = [] for target in targets: # Encode the gRNA sequence encoded_seq = get_seqcode(target[0]).reshape(-1,4,1,23) # Predict on-target efficiency using the model prediction = dcModel.ontar_predict(encoded_seq) # Format output gRNA = target[1] chr = target[2] start = target[3] end = target[4] strand = target[5] formatted_data.append([chr, start, end, strand, target[0], gRNA, prediction[0]]) return formatted_data def fetch_ensembl_transcripts(gene_symbol): url = f"https://rest.ensembl.org/lookup/symbol/homo_sapiens/{gene_symbol}?expand=1;content-type=application/json" response = requests.get(url) if response.status_code == 200: gene_data = response.json() if 'Transcript' in gene_data: return gene_data['Transcript'] else: print("No transcripts found for gene:", gene_symbol) return None else: print(f"Error fetching gene data from Ensembl: {response.text}") return None def fetch_ensembl_sequence(transcript_id): url = f"https://rest.ensembl.org/sequence/id/{transcript_id}?content-type=application/json" response = requests.get(url) if response.status_code == 200: sequence_data = response.json() if 'seq' in sequence_data: return sequence_data['seq'] else: print("No sequence found for transcript:", transcript_id) return None else: print(f"Error fetching sequence data from Ensembl: {response.text}") return None def find_crispr_targets(sequence, chr, start, strand, pam="NGG", target_length=20): targets = [] len_sequence = len(sequence) for i in range(len_sequence - len(pam) + 1): if sequence[i + 1:i + 3] == pam[1:]: if i >= target_length: target_seq = sequence[i - target_length:i + 3] tar_start = start + i - target_length tar_end = start + i + 3 gRNA = sequence[i - target_length:i] targets.append([target_seq, gRNA, chr, str(tar_start), str(tar_end), str(strand)]) return targets def process_gene(gene_symbol, model_path): transcripts = fetch_ensembl_transcripts(gene_symbol) all_data = [] gene_sequence = '' # Initialize an empty string for the gene sequence if transcripts: for transcript in transcripts: transcript_id = transcript['id'] chr = transcript.get('seq_region_name', 'unknown') start = transcript.get('start', 0) strand = transcript.get('strand', 'unknown') # Fetch the sequence here and concatenate if multiple transcripts gene_sequence += fetch_ensembl_sequence(transcript_id) or '' # Fetch exon and CDS information exons = fetch_ensembl_exons(transcript_id) cds_list = fetch_ensembl_cds(transcript_id) # You might want to do something specific with exons and CDS information here # For example, store them, print them, or include them in your analysis if gene_sequence: gRNA_sites = find_crispr_targets(gene_sequence, chr, start, strand) if gRNA_sites: formatted_data = format_prediction_output(gRNA_sites, model_path) all_data.extend(formatted_data) # Return the data, fetched sequence, and possibly exon/CDS data return all_data, gene_sequence, exons, cds_list def fetch_ensembl_exons(transcript_id): """Fetch exon information for a given transcript from Ensembl.""" url = f"https://rest.ensembl.org/overlap/id/{transcript_id}?feature=exon;content-type=application/json" response = requests.get(url) if response.status_code == 200: return response.json() # Returns a list of exons for the transcript else: print(f"Error fetching exon data from Ensembl for transcript {transcript_id}: {response.text}") return None def fetch_ensembl_cds(transcript_id): """Fetch coding sequence (CDS) information for a given transcript from Ensembl.""" url = f"https://rest.ensembl.org/overlap/id/{transcript_id}?feature=cds;content-type=application/json" response = requests.get(url) if response.status_code == 200: return response.json() # Returns a list of CDS regions for the transcript else: print(f"Error fetching CDS data from Ensembl for transcript {transcript_id}: {response.text}") return None def create_genbank_features(formatted_data): features = [] for data in formatted_data: # Strand conversion to Biopython's convention strand = 1 if data[3] == '+' else -1 location = FeatureLocation(start=int(data[1]), end=int(data[2]), strand=strand) feature = SeqFeature(location=location, type="misc_feature", qualifiers={ 'label': data[5], # Use gRNA as the label 'target': data[4], # Include the target sequence 'note': f"Prediction: {data[6]}" # Include the prediction score }) features.append(feature) return features def generate_genbank_file_from_df(df, gene_sequence, gene_symbol, output_path): features = [] for index, row in df.iterrows(): location = FeatureLocation(start=int(row["Start Pos"]), end=int(row["End Pos"]), strand=int(row["Strand"])) feature = SeqFeature(location=location, type="gene", qualifiers={ 'locus_tag': row["Gene ID"], # Assuming Gene ID is equivalent to Chromosome here 'note': f"gRNA: {row['gRNA']}, Prediction: {row['Prediction']}" }) features.append(feature) record = SeqRecord(Seq(gene_sequence), id=gene_symbol, name=gene_symbol, description='CRISPR Cas9 predicted targets', features=features) # Add the missing molecule_type annotation record.annotations["molecule_type"] = "DNA" SeqIO.write(record, output_path, "genbank") def create_bed_file_from_df(df, output_path): with open(output_path, 'w') as bed_file: for index, row in df.iterrows(): chrom = row["Gene ID"] start = int(row["Start Pos"]) end = int(row["End Pos"]) strand = '+' if row["Strand"] == '+' else '-' gRNA = row["gRNA"] score = str(row["Prediction"]) # Ensure score is converted to string if not already bed_file.write(f"{chrom}\t{start}\t{end}\t{gRNA}\t{score}\t{strand}\n") def create_csv_from_df(df, output_path): df.to_csv(output_path, index=False)