ࡱ>  ~&bjbjww .J^k^kFT   8<UT*^(*******=-/d***###*#*##:),)P F) n**0*P)RW0"W0)W0)#**#j*W0 }: TrAEL-seq adaptor testing Not all adaptors work unfortunately, and we have had multiple dud batches. In this protocol, all the multiplex adaptors you want to test (usually 6) are mixed and ligated to a single pre-adenylated substrate, then PCRd up and sequenced by Plasmidsaurus. A python script then extracts the frequency of reads coming from the different barcodes. This is the validation oligo (it needs to be synthesised as an RNA oligo by Merck, but it is a DNA oligo with 2 rA nucleotides on the end) [dA][dG][dA][dC][dG][dT][dG][dT][dG][dC][dT][dC][dT][dT][dC][dC][dG][dA][dT][dC][dT]AA 2ul 10x T4 RNA ligase buffer 0.2ul 10uM TrAEL-validation oligo (ie: 1:10 dilution of stock) 0.2ul each pre-adenylated adaptor to be tested 8ul 50% PEG Water to 19.5ul, vortex very well Add 0.5ul T4 RNA ligase KQ Mix by pipetting Incubate overnight at 25 Add 1ul 10mM dNTPs 0.5ul Bst2 Warmstart Mix by pipetting Incubate 15 min at 65 Purify down Monarch column USING OLIGO PROTOCOL, elute in 10ul Amplify using normal NEB next primers and NEBNext PCR mix, 25 cycles 10uL of DNA eluate 1uL NEBNext mixed indexes 1uL NEBNext universal PCR primer 12.5uL UltraII Q5 PCR mix 0.5uL nuclease free water Purify down Monarch column USING DNA PROTOCOL, elute in 15ul Sequence the product on Plasmidsaurus Quantitate DNA with Nanodrop (make sure this is at least 1ng/uL) Place order on Plasmidsaurus website (Standard Premium PCR for 3,000 reads) and print order QR code Transfer 10uL of purified DNA to PCR strip tubes and label following Plasmidsaurus instructions (see below). Place samples and order QR code in drop box by Stores  Volume and concentration of sample needed based on sequencing depth  Download the fastq file and run this python script on it (script is on the next page): Python3 analyse_TrAEL_adaptors.py FASTQ-FILE Sample Output: Barcode Frequency (%) Position of ligated sequence Frequency of Position (%) Barcode 7 30.77 3 73.17 Barcode 6 25.54 3 63.00 Barcode 5 16.89 3 63.62 Barcode 1 16.40 3 59.53 Barcode 8 3.48 3 8.08 Barcode 3 2.81 3 56.25 tgct 1.19 2 79.41 gtgt 1.12 2 15.62 aagt 0.91 4 23.08 aggt 0.88 2 64.00 Script finds the 10 most frequent barcodes, and gives their percentage frequency amongst those top 10. It identifies the ones that it knows (barcodes 1,3,5,6,7,8). In this example data you can see that adaptors 8 and 3 do not work well and shouldnt be used. The difference between 1 and 7 is probably not too big an issue remember that in a real experiment the adaptors are not competing. It then looks for the position at which the ligated DNA is most frequently found relative to the barcode this SHOULD be at position 3. Then it gives the frequency of reads with that barcode where the ligated sequence is found at that position this should be >50% if not higher. In this case, Adaptor Barcode 8 has a load of heterogenous shit after the barcode and before the ligation junction so that only 8% of ligations are actually in the correct place. #!/usr/bin/env python3 from collections import Counter, defaultdict import argparse # Target sequences ANCHOR_SEQ = "CACTCTTTCCCTACACGACGCTCTTCCGATCT" DOWNSTREAM_SEQ = "GATCGGA" # Barcode settings BARCODE_OFFSET = 8 BARCODE_LENGTH = 4 # Barcode name mapping BARCODE_NAMES = { "agtc": "Barcode 1", "cttg": "Barcode 3", "aagg": "Barcode 5", "ttcc": "Barcode 6", "gtgc": "Barcode 7", "gcca": "Barcode 8", } def reverse_complement(seq): """Generate reverse complement of DNA sequence.""" complement = str.maketrans("ACGTNacgtn", "TGCANtgcan") return seq.translate(complement)[::-1] def read_fastq_sequences(fastq_file): """ Read FASTQ file and extract sequence lines (line 2 and every 4th line thereafter). """ sequences = [] with open(fastq_file, "r") as f: for i, line in enumerate(f): if i % 4 == 1: sequences.append(line.strip()) return sequences def generate_all_sequences(sequences): """Add reverse complements to sequence list.""" revcomp_sequences = [reverse_complement(seq) for seq in sequences] return sequences + revcomp_sequences def extract_barcode_and_position(seq): """ Find anchor sequence. Extract 4-nt barcode beginning 9 nt after anchor end. Find position of GATCGGA relative to barcode end. """ anchor_pos = seq.find(ANCHOR_SEQ) if anchor_pos == -1: return None, None anchor_end = anchor_pos + len(ANCHOR_SEQ) barcode_start = anchor_end + BARCODE_OFFSET barcode_end = barcode_start + BARCODE_LENGTH if barcode_end > len(seq): return None, None barcode = seq[barcode_start:barcode_end].lower() downstream_pos = seq.find(DOWNSTREAM_SEQ, barcode_end) if downstream_pos == -1: return barcode, None relative_position = downstream_pos - barcode_end return barcode, relative_position def barcode_label(barcode): """ Return named barcode if present in lookup table, otherwise return barcode sequence itself. """ return BARCODE_NAMES.get(barcode.lower(), barcode) def main(): parser = argparse.ArgumentParser( description="Extract 4-nt barcodes from FASTQ reads and analyze GATCGGA positions." ) parser.add_argument( "fastq", help="Input FASTQ file" ) args = parser.parse_args() print("Reading FASTQ sequences...") sequences = read_fastq_sequences(args.fastq) print(f"Loaded {len(sequences)} sequences") print("Generating reverse complements...") all_sequences = generate_all_sequences(sequences) print(f"Total sequences including reverse complements: {len(all_sequences)}") barcode_counter = Counter() barcode_positions = defaultdict(list) print("Searching for anchor sequence and extracting barcodes...") for seq in all_sequences: barcode, rel_pos = extract_barcode_and_position(seq) if barcode is not None: barcode_counter[barcode] += 1 if rel_pos is not None: barcode_positions[barcode].append(rel_pos) # Top 10 barcodes top10 = barcode_counter.most_common(10) # Total reads among top 10 barcodes total_top10_reads = sum(count for barcode, count in top10) print() print(f"Total reads containing top 10 barcodes:\t{total_top10_reads}") print() # Output table header print("Barcode\tFrequency (%)\tPosition of ligated sequence\tFrequency of Position (%)") for barcode, count in top10: barcode_name = barcode_label(barcode) # Barcode percentage among top 10 reads barcode_percent = (count / total_top10_reads) * 100 positions = barcode_positions[barcode] if len(positions) > 0: pos_counter = Counter(positions) most_common_position, pos_freq = pos_counter.most_common(1)[0] # Percentage within reads containing this barcode pos_percent = (pos_freq / count) * 100 print( f"{barcode_name}\t" f"{barcode_percent:.2f}\t" f"{most_common_position}\t" f"{pos_percent:.2f}" ) else: print( f"{barcode_name}\t" f"{barcode_percent:.2f}\t" f"NA\t" f"0.00" ) if __name__ == "__main__": main()  If more reads are needed, see table below and adjust sample volume      FILENAME TrAEL-seq adaptor testing v1.0 Houseley lab  PAGE 1 r s t V W X u   . ? 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