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File formats

File formats#

Input file formats for Casanovo#

When you’re ready to use Casanovo for de novo peptide sequencing, you can input your MS/MS spectra in one of the following formats:

  • mzML: XML-based mass spectrometry community standard file format developed by the Proteomics Standards Initiative (PSI).

  • mzXML: XML-based predecessor of mzML. Although supported by Casanovo, mzML should typically be preferred instead.

  • MGF: A simple text-based peak file, though not as rich in detail as mzML.

All three of the above file formats can be used as input to Casanovo for de novo peptide sequencing. As the official PSI standard format containing the complete information from a mass spectrometry run, mzML should typically be preferred.

Output: Understanding the mzTab format#

After Casanovo processes your input file(s), it provides the sequencing results in an mzTab file. This file is divided into two main sections:

  1. Metadata section: This part describes general information about the file and the Casanovo sequencing task.

  2. Peptide–spectrum match (PSM) section: Details of the peptide sequences that Casanovo predicted for the MS/MS spectra.

mzTab files can contain additional sections to include protein identifications and quantification information as well. However, as these levels of information are not relevant for Casanovo, these are not included in its output mzTab files.

Tip

mzTab is a human and machine readable format. It can be inspected manually by opening it with a text editor or with spreadsheet software (specify tab as the delimiter). Additionally, you can use tools like Pyteomics for Python or MSnbase for R to programmatically read mzTab files.

Metadata section

The metadata section consists of three columns, each separated by a tab:

  1. The prefix MTD indicating that this is the metadata section.

  2. A key describing a metadata item.

  3. The value corresponding to the metadata key.

As an example, these are the first few lines in an mzTab output file produced by Casanovo:

MTD	mzTab-version	1.0.0
MTD	mzTab-mode	Summary
MTD	mzTab-type	Identification
MTD	description	Casanovo identification file my_example_output
MTD	software[1]	[MS, MS:1003281, Casanovo, 4.0.1]

This identifies this mzTab file with filename “my_example_output” as a summary-level identification file produced by Casanovo. On the final line you can see a typical key–value entry using information defined in the PSI-MS controlled vocabulary. In this case, the line indicates that the file is produced by the Casanovo software, which is recorded in the MS controlled vocabulary with accession number MS:1003281. The final element is the version number of Casanovo that produced this file.

The next few lines typically list the post-translational modifications (PTMs) that Casanovo knew:

MTD	fixed_mod[1]	[UNIMOD, UNIMOD:4, Carbamidomethyl, ]
MTD	fixed_mod[1]-site	C
MTD	variable_mod[1]	[UNIMOD, UNIMOD:7, Deamidated, ]
MTD	variable_mod[1]-site	N
MTD	variable_mod[2]	[UNIMOD, UNIMOD:7, Deamidated, ]
MTD	variable_mod[2]-site	Q
MTD	variable_mod[3]	[UNIMOD, UNIMOD:35, Oxidation, ]
MTD	variable_mod[3]-site	M
MTD	variable_mod[4]	[UNIMOD, UNIMOD:385, Ammonia-loss, ]
MTD	variable_mod[4]-site	N-term
MTD	variable_mod[5]	[UNIMOD, UNIMOD:1, Acetyl, ]
MTD	variable_mod[5]-site	N-term
MTD	variable_mod[6]	[UNIMOD, UNIMOD:5, Carbamyl, ]
MTD	variable_mod[6]-site	N-term

This indicates that cysteine carbamidomethylation was used as a static modification (this time defined by the Unimod controlled vocabulary), and that deamidation of asparagine and glutamine, oxidation of methionine, N-terminal loss of ammonia, N-terminal acetylation, and N-terminal carbamylation were used as variable modifications. Different PTMs in Casanovo can only be enabled or disabled by training a new model.

The final piece of information in the metadata section is the active configuration settings, allowing for replication or review of the analysis parameters:

MTD	software[1]-setting[1]	model = casanovo_massivekb_v4_0_0.ckpt
MTD	software[1]-setting[2]	config_filename = default
MTD	software[1]-setting[3]	precursor_mass_tol = 50.0
MTD	software[1]-setting[4]	isotope_error_range = (0, 1)
MTD	software[1]-setting[5]	min_peptide_len = 6
MTD	software[1]-setting[6]	predict_batch_size = 1024
MTD	software[1]-setting[7]	n_beams = 10
MTD	software[1]-setting[8]	top_match = 1
MTD	software[1]-setting[9]	accelerator = auto
MTD	software[1]-setting[10]	devices = None
MTD	software[1]-setting[11]	random_seed = 454
MTD	software[1]-setting[12]	n_log = 1
MTD	software[1]-setting[13]	tb_summarywriter = None
MTD	software[1]-setting[14]	save_top_k = 5
MTD	software[1]-setting[15]	model_save_folder_path =
MTD	software[1]-setting[16]	val_check_interval = 50000
MTD	software[1]-setting[17]	n_peaks = 150
MTD	software[1]-setting[18]	min_mz = 50.0
MTD	software[1]-setting[19]	max_mz = 2500.0
MTD	software[1]-setting[20]	min_intensity = 0.01
MTD	software[1]-setting[21]	remove_precursor_tol = 2.0
MTD	software[1]-setting[22]	max_charge = 10
MTD	software[1]-setting[23]	dim_model = 512
MTD	software[1]-setting[24]	n_head = 8
MTD	software[1]-setting[25]	dim_feedforward = 1024
MTD	software[1]-setting[26]	n_layers = 9
MTD	software[1]-setting[27]	dropout = 0.0
MTD	software[1]-setting[28]	dim_intensity = None
MTD	software[1]-setting[29]	max_length = 100
MTD	software[1]-setting[30]	warmup_iters = 100000
MTD	software[1]-setting[31]	max_iters = 600000
MTD	software[1]-setting[32]	learning_rate = 0.0005
MTD	software[1]-setting[33]	weight_decay = 1e-05
MTD	software[1]-setting[34]	train_label_smoothing = 0.01
MTD	software[1]-setting[35]	train_batch_size = 32
MTD	software[1]-setting[36]	max_epochs = 30
MTD	software[1]-setting[37]	num_sanity_val_steps = 0
MTD	software[1]-setting[38]	train_from_scratch = True
MTD	software[1]-setting[39]	calculate_precision = False
MTD	software[1]-setting[41]	n_workers = 20
MTD	ms_run[1]-location	file://[...]/my_example_input.mgf

PSM section

The PSM section in mzTab files starts with a header line, indicated by the PSH key, which defines the subsequent tabular PSM information. Next, the following lines each start with the PSM key and contain information for an individual PSM predicted by Casanovo.

PSH	sequence	PSM_ID	accession	unique	database	database_version	search_engine	search_engine_score[1]	modifications	retention_time	charge	exp_mass_to_charge	calc_mass_to_charge	spectra_ref	pre	post	start	end	opt_ms_run[1]_aa_scores
PSM	EPPTPLTYVAGAGSGVR	1	null	null	null	null	[MS, MS:1003281, Casanovo, 4.0.1]	0.968312939008077	null	null	2.0	836.439	836.4386613168799	ms_run[1]:index=0	null	null	null	null	0.96454,0.90841,0.97874,0.97979,0.97915,0.98254,0.98184,0.97898,0.86762,0.97782,0.97771,0.97899,0.97987,0.97788,0.97949,0.98074,0.97561
PSM	VVHGFYNPAVSRVLEQ	2	null	null	null	null	[MS, MS:1003281, Casanovo, 4.0.1]	0.9652494998539195	null	null	3.0	605.6572	605.65644936688	ms_run[1]:index=1	null	null	null	null	0.96870,0.97701,0.85667,0.97274,0.97827,0.97790,0.97829,0.97706,0.97654,0.97725,0.97778,0.95544,0.95622,0.96240,0.96992,0.96909
PSM	EPPTPLTYVAGGSLNR	3	null	null	null	null	[MS, MS:1003281, Casanovo, 4.0.1]	0.813004752730622	null	null	2.0	836.4398	836.4386608168799	ms_run[1]:index=2	null	null	null	null	0.78636,0.45168,0.64947,0.68432,0.89344,0.90091,0.90124,0.56938,0.89757,0.90204,0.90129,0.90190,0.80076,0.90097,0.90233,0.87599
PSM	LERPFVHLM+15.995VFLGGSGR	4	null	null	null	null	[MS, MS:1003281, Casanovo, 4.0.1]	0.758128507890635	null	null	4.0	483.7627	483.51345739187997	ms_run[1]:index=3	null	null	null	null	0.86884,0.85508,0.87392,0.39732,0.87556,0.87291,0.69642,0.87083,0.79858,0.86588,0.86291,0.84178,0.45706,0.52835,0.85704,0.41526,0.83419
PSM	GEYKLLPFNKLMLGEG	5	null	null	null	null	[MS, MS:1003281, Casanovo, 4.0.1]	-0.18260370983796959	null	null	3.0	602.99817	603.6586910335465	ms_run[1]:index=4	null	null	null	null	0.64453,0.77152,0.90248,0.84100,0.65059,0.89975,0.87886,0.82220,0.90324,0.90056,0.88657,0.86091,0.66978,0.63579,0.81815,0.90577
...

Key information for each PSM is as follows:

  • sequence: The predicted peptide sequence.

  • PSM_ID: A monotonically increasing index, serving as a unique identifier for each PSM.

  • search_engine_score[1]: The score of this PSM.

  • spectra_ref: Unique identifier linking the prediction back to the original spectrum in the input file(s).

  • opt_ms_run[1]_aa_scores: Casanovo predicts peptides in an autoregressive fashion, one amino acid at a time. This column contains comma-separated scores of the individual amino acid predictions.

Note

Scores in Casanovo range from -1 to 1, where 1 indicates high confidence in the prediction. A score below 0 occurs for a predicted peptide sequence that mismatches the observed precursor mass, in which case the score is penalized by subtracting 1. This will also be evident from a difference in the observed precursor m/z, in the exp_mass_to_charge column, and the precursor m/z calculated from the predicted peptide sequence, in the calc_mass_to_charge column. Hence, it is important to properly configure settings that impact the precursor mass filter, such as the precursor mass tolerance (option precursor_mass_tol) and the isotopes to consider (option isotope_error_range).

The spectra_ref column is essential for connecting predictions back to the corresponding MS/MS spectra in the input file(s). This column consists of two parts: the run index and the spectrum reference, separated by a colon.

  • The run index is of the form ms_run[FILE_INDEX], with FILE_INDEX referring to the corresponding run location in the metadata section. In the typical case when only a single input file was processed, this will be 1.

  • The spectrum reference can take the form of either a scan number or a spectrum index.

    • When using mzML or mzXML files as input, the spectrum reference will take the form of a scan number, encoded as scan=SCAN, with SCAN the scan number specified in the input file for this spectrum.

    • When using MGF files as input, the spectrum reference will be an index, encoded as index=INDEX, with INDEX the zero-based index of the spectrum in its input file. This is because MGF is not a standardized format that is not guaranteed to contain specific spectrum identifiers.

Warning

Be mindful of the input peak file format when linking Casanovo PSMs to their input spectra. Even when the same raw file is converted to both mzML and MGF, scan numbers in the mzML file will generally not match spectrum indices in the MGF file, as the former contains both MS and MS/MS spectra while the latter only contains MS/MS spectra.

Note

The PSM identifier in the PSM_ID column is not necessarily identical to the spectrum index in the spectra_ref column, even for MGF files.

  • PSM_ID is one-based, whereas spectrum indices in spectra_ref are zero-based.

  • If multiple predictions are included per spectrum (configuration option top_match), each PSM will have a different identifier, but spectrum references will overlap.

Casanovo configuration#

Casanovo operates based on settings defined in a YAML configuration file. This file contains several options that affect how Casanovo processes your data and predicts peptide sequences. If you run Casanovo without specifying a configuration file, it uses a set of default settings. However, you might want to adjust these settings for several reasons, such as to capture specific characteristics of your data or to experiment with different training configurations.

To create a custom configuration file, you can start by generating a copy of the default configuration:

casanovo configure

You can then edit this file to adjust various settings. After editing, specify your custom configuration file when running Casanovo with the --config option.

The configuration file is divided into sections, each containing options that are relevant to different phases of Casanovo’s operation. The first section contains options used to configure Casanovo during de novo peptide sequencing, followed by options in the second section that can only be modified when training a new model. For example, the top_match option in the first section makes it possible to flexibly report multiple PSMs per spectrum during de novo peptide sequencing. In contrast, setting a different value for the n_peaks option in the second section is only possible when training a new model, and cannot be modified when predicting with a previously trained model that uses a different configuration.

Tip

Each change in the configuration can lead to different outcomes in the peptide sequencing process, so it may be beneficial to experiment with various settings to find the optimal configuration for your data. Always consider your experimental design and the nature of your data when adjusting these settings.

Logging#

Casanovo generates detailed log files during operation, providing insights into its performance and aiding in troubleshooting. These log files are named similarly to the output mzTab files but with a .log extension. Log files detail every step Casanovo takes, including:

  • Starting and ending timestamps of the sequencing or training process.

  • Configuration options used.

  • Warnings or errors encountered during processing, providing clues for troubleshooting.

  • Summary statistics upon completion, offering a quick overview of the results.

Tip

Tips for using log files:

  • Bug reporting: When encountering issues, including the relevant log file in your bug report can significantly aid in diagnosing the problem.

  • Performance monitoring: Log files can be used to monitor the efficiency of Casanovo’s operation over time, identifying potential bottlenecks.

For advanced users: Training Casanovo#

To train a new Casanovo model, the training and validation data must be provided as annotated MGF files. Annotated MGF files are similar to standard MGF files but include a SEQ key–value pair in the spectrum header, indicating the peptide sequence for the corresponding spectrum.

Example of an annotated MGF file entry:

BEGIN IONS
TITLE=My spectrum title
PEPMASS=602.2881
CHARGE=2+
RTINSECONDS=985.44604
SEQ=HQGVM+15.995VGM+15.995GQK
84.08081817626953 0.1001848503947258
87.74003601074219 0.07622149586677551
...
END IONS

Note

In case the peptide sequence includes PTMs, ensure that these are formatted correctly and match the amino acid and modification vocabulary in the Casanovo configuration.

mzML or mzXML files are not supported as input during training, as these formats do not provide a mechanism to annotate their spectra with peptide sequences. Similarly, in Casanovo evaluation mode only annotated MGF files are supported.

During training, Casanovo will save checkpoint files at every val_check_interval steps, specified in the configuration. Model checkpoints will be saved in the model_save_folder_path folder with filename format epoch=EPOCH-step=STEP.ckpt, with EPOCH the epoch and STEP the training step at which the checkpoint was taken, helping you track progress and select the best model based on validation performance.

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