From a1d1e9a2f9475c7db60f80834a6ddf2dd1fb5923 Mon Sep 17 00:00:00 2001 From: Psy-Fer Date: Thu, 27 Oct 2022 17:20:14 +1100 Subject: [PATCH] add slow5 reading --- radian/basecall.py | 211 +++++++++++++++++++++++----------- radian/data/fast5/reads.fast5 | Bin 0 -> 352198 bytes radian/data/reads.blow5 | Bin 0 -> 52687 bytes requirements.txt | 1 + 4 files changed, 145 insertions(+), 67 deletions(-) create mode 100644 radian/data/fast5/reads.fast5 create mode 100644 radian/data/reads.blow5 diff --git a/radian/basecall.py b/radian/basecall.py index ae7595f..6dee5a1 100644 --- a/radian/basecall.py +++ b/radian/basecall.py @@ -5,6 +5,7 @@ import numpy as np from ont_fast5_api.fast5_interface import get_fast5_file +import pyslow5 from decode import beam_search from matrix_assembly import assemble_matrices, plot_assembly @@ -67,75 +68,151 @@ def main(): fasta = open(f"{args.fasta_dir}/reads-{fasta_n}.fasta", "w") # Basecall each read in fast5 directory - for fast5_filepath in Path(args.fast5_dir).rglob('*.fast5'): - with get_fast5_file(fast5_filepath, 'r') as fast5: - for read in fast5.get_reads(): - start_t = time() - - # Preprocess read - raw_signal = read.get_raw_data() - try: - norm_signal = mad_normalise(raw_signal, args.outlier_clip) - except ValueError as e: - print(e.args) - print(f"{read.read_id} signal issue, skipping this read.") - continue - windows, pad = get_windows(norm_signal, args.chunk_len, args.step_size) - - # Pass windows through signal model in batches - i = 0 - matrices = [] - while i + args.batch_size <= len(windows): - batch = windows[i:i+args.batch_size] - i += args.batch_size - matrices.extend(sig_model.predict(batch)) - if i < len(windows): - matrices.extend(sig_model.predict(windows[i:])) - - # Trim padding from last matrix before decoding - matrices[-1] = matrices[-1][:-pad] - - # Decode CTC output (with/without RNA model, global/local) - if args.decode_type == "global": - matrix = assemble_matrices(matrices, args.step_size) - # plot_assembly(matrices, matrix, args.chunk_len, args.step_size) + # this is a hack, just detecting the .blow5 extention to hijack the arg + # should do something better than this + if args.fast5_dir.split(".")[-1] == "blow5": + SLOW5 = True + s5 = pyslow5.Open(args.fast5_dir, 'r') + # this can be multithreaded with s5.seq_reads_multi(), see docs for flags + reads = s5.seq_reads() + # this is copy/paste of below but using read_id set first rather than accessing it + for read in reads: + start_t = time() + read_id = read["read_id"] + # Preprocess read + raw_signal = read["signal"] + try: + norm_signal = mad_normalise(raw_signal, args.outlier_clip) + except ValueError as e: + print(e.args) + print(f"{read_id} signal issue, skipping this read.") + continue + windows, pad = get_windows(norm_signal, args.chunk_len, args.step_size) + + # Pass windows through signal model in batches + i = 0 + matrices = [] + while i + args.batch_size <= len(windows): + batch = windows[i:i+args.batch_size] + i += args.batch_size + matrices.extend(sig_model.predict(batch)) + if i < len(windows): + matrices.extend(sig_model.predict(windows[i:])) + + # Trim padding from last matrix before decoding + matrices[-1] = matrices[-1][:-pad] + + # Decode CTC output (with/without RNA model, global/local) + if args.decode_type == "global": + matrix = assemble_matrices(matrices, args.step_size) + # plot_assembly(matrices, matrix, args.chunk_len, args.step_size) + sequence = beam_search(matrix, + 'ACGT', + args.beam_width, + rna_model, + args.sig_threshold, + args.rna_threshold, + args.context_len, + entropy_cache) + else: + read_fragments = [] + for matrix in matrices: sequence = beam_search(matrix, - 'ACGT', - args.beam_width, - rna_model, - args.sig_threshold, - args.rna_threshold, - args.context_len, - entropy_cache) - else: - read_fragments = [] - for matrix in matrices: + 'ACGT', + args.beam_width, + None, + None, + None, + None, + None) + read_fragments.append(sequence) + consensus = simple_assembly(read_fragments) + sequence = index2base(np.argmax(consensus, axis=0)) + + end_t = time() + dur = end_t - start_t + + # Write read to fasta file (reverse sequence to be 5' to 3') + fasta.write(f">{read_id}\n{sequence[::-1]}\n") + fasta_i += 1 + print(f"Basecalled read {read_id} in {dur:.2f} sec.") + + # Only write 1000 reads per fasta file + if fasta_i == 1000: + fasta.close() + fasta_n += 1 + fasta = open(f"{args.fasta_dir}/reads-{fasta_n}.fasta", "w") + fasta_i = 0 + else: + for fast5_filepath in Path(args.fast5_dir).rglob('*.fast5'): + with get_fast5_file(fast5_filepath, 'r') as fast5: + for read in fast5.get_reads(): + start_t = time() + + # Preprocess read + raw_signal = read.get_raw_data() + try: + norm_signal = mad_normalise(raw_signal, args.outlier_clip) + except ValueError as e: + print(e.args) + print(f"{read.read_id} signal issue, skipping this read.") + continue + windows, pad = get_windows(norm_signal, args.chunk_len, args.step_size) + + # Pass windows through signal model in batches + i = 0 + matrices = [] + while i + args.batch_size <= len(windows): + batch = windows[i:i+args.batch_size] + i += args.batch_size + matrices.extend(sig_model.predict(batch)) + if i < len(windows): + matrices.extend(sig_model.predict(windows[i:])) + + # Trim padding from last matrix before decoding + matrices[-1] = matrices[-1][:-pad] + + # Decode CTC output (with/without RNA model, global/local) + if args.decode_type == "global": + matrix = assemble_matrices(matrices, args.step_size) + # plot_assembly(matrices, matrix, args.chunk_len, args.step_size) sequence = beam_search(matrix, - 'ACGT', - args.beam_width, - None, - None, - None, - None, - None) - read_fragments.append(sequence) - consensus = simple_assembly(read_fragments) - sequence = index2base(np.argmax(consensus, axis=0)) - - end_t = time() - dur = end_t - start_t - - # Write read to fasta file (reverse sequence to be 5' to 3') - fasta.write(f">{read.read_id}\n{sequence[::-1]}\n") - fasta_i += 1 - print(f"Basecalled read {read.read_id} in {dur:.2f} sec.") - - # Only write 1000 reads per fasta file - if fasta_i == 1000: - fasta.close() - fasta_n += 1 - fasta = open(f"{args.fasta_dir}/reads-{fasta_n}.fasta", "w") - fasta_i = 0 + 'ACGT', + args.beam_width, + rna_model, + args.sig_threshold, + args.rna_threshold, + args.context_len, + entropy_cache) + else: + read_fragments = [] + for matrix in matrices: + sequence = beam_search(matrix, + 'ACGT', + args.beam_width, + None, + None, + None, + None, + None) + read_fragments.append(sequence) + consensus = simple_assembly(read_fragments) + sequence = index2base(np.argmax(consensus, axis=0)) + + end_t = time() + dur = end_t - start_t + + # Write read to fasta file (reverse sequence to be 5' to 3') + fasta.write(f">{read.read_id}\n{sequence[::-1]}\n") + fasta_i += 1 + print(f"Basecalled read {read.read_id} in {dur:.2f} sec.") + + # Only write 1000 reads per fasta file + if fasta_i == 1000: + fasta.close() + fasta_n += 1 + fasta = open(f"{args.fasta_dir}/reads-{fasta_n}.fasta", "w") + fasta_i = 0 # Make sure last fasta file is closed fasta.close() diff --git a/radian/data/fast5/reads.fast5 b/radian/data/fast5/reads.fast5 new file mode 100644 index 0000000000000000000000000000000000000000..1d3ae7f092b75dc4cb04964823c8956083749963 GIT binary patch literal 352198 zcmeFacXU_9xA&hjC%uu75Rw2P1V{oQA%uhk2oMON_g;h$dT#=v2#O7{qF=?{P!Rq4 zMFkNB6r_uySOF12@4fdtulIbCdw<`%?p?q2{PV2!tcQHo$vJ0c&z?Pd_UzfSr<}QM zTDA%aj1BbCpTEDCw^x{bEc;hd`nRdj#|D+Xm&Fe%eQ-+ePnO;n8_$CSM#V*!hKH5n zk9R!*50{4b=vGkR1!NiivU_Q?*C1cd)2n3xcklmCaiC2>^DZjzpi)7*R$BUQ!#slY zKgn6xKu>})x%}T{T6n3vmZdL~>tL@oNVX(4#4FOv-)s1&vBL_d4V!%Ls0rhhM~vgx zlxj^L3j7sZ`t0RpsrCewhW&fHv%nVqzi&Tf4XTW;|G)P0YJrD}^fJVE@BdG6psfA$ zDJ^wg>D|9nua+^RThV>?amdJ`@#BY$EgUt}6EkGO_$kAtPbr*IG~!-`OqpCXWX!1X zBgz0un`N0;dJ^%!d#BDV9WNhRi0AL!QoUaWzoqp3n5TR38d(ar-Ouw-2Dg2^r$afn z(R1HY3ioPh`!0iv>#A1i<<+#i=blpvxA?!{o8I4(;?)7A_fohm|Iu%G0#*+85ct>7 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