Configuration

There are a lot of parameters allowing to customize the pipeline. They can be grouped into several categories. The parameters will be displayed when running splash without parameters (or with --help).

Input

• input_file — positional argument, path to the file where input samples are defined, the format is: per each line {sample_name} {path} path is a fastq[.gz] file in case of non-10X and txt file for 10X/Visium where the content of text file is {first_file.fastq[.gz]},{second_file.fastq[.gz]} per line

Base configuration

• --outname_prefix — prefix of output file names (default: result)
• --anchor_len — anchor length (default: 31)
• --gap_len — gap length, if 'auto' it will be inferred from the data, in the opposite case it must be an int (default: 0)
• --target_len — target length (default: 31)
• --anchor_list — list of accepted anchors, this is path to plain text file with one anchor per line without any header (default accept all anchors)
• --pvals_correction_col_name — for which column correction should be applied (default: pval_opt)
• --technology — Technology used to generate the input data, must be one of 'base', '10x', 'visium' (default: base)
• --without_compactors — if used compactors will not be run (default: False)
• --compactors_config — optional json file with compactors configuration, example file content: { "num_threads": 4, "epsilon": 0.001 } (default: )
• --lookup_table_config — optional json file with configuration of lookup_table, if not specified lookup_tables are not used (default: )

Filters and thresholds

• --poly_ACGT_len — filter out all anchors containing poly(ACGT) of length at least <poly_ACGT_len> (0 means no filtering) (default: 8)
• --artifacts — path to artifacts, each anchor containing artifact will be filtered out (default: )
• --dont_filter_illumina_adapters — if used anchors containing Illumina adapters will not be filtered out (default: False)
• --anchor_unique_targets_threshold — filter out all anchors for which the number of unique targets is <= anchor_unique_targets_threshold (default: 1)
• --anchor_count_threshold — filter out all anchors for which the total count <= anchor_count_threshold (default: 50)
• --anchor_samples_threshold — filter out all anchors for which the number of unique samples is <= anchor_samples_threshold (default: 1)
• --anchor_sample_counts_threshold — filter out anchor from sample if its count in this sample is <= anchor_sample_counts_threshold (default: 5)
• --n_most_freq_targets_for_stats — use at most n_most_freq_targets_for_stats for each contingency table, 0 means use all (default: 0)
• --n_most_freq_targets_for_dump — use when dumping satc (txt or binary), resulting file will only contain data for n_most_freq_targets_for_dump targets in each anchor, 0 means use all (default: 0)
• --fdr_threshold — keep anchors having corrected p-val below this value (default: 0.05)
• --min_hamming_threshold — keep only anchors with a pair of targets that differ by >= min_hamming_threshold (default: 0)
• --keep_top_n_target_entropy — select keep_top_n_target_entropy records with highest target entropy (0 means don't select) (default: 10000)
• --keep_top_n_effect_size_bin — select keep_top_n_effect_size_bin records with highest effect size bin (0 means don't select) (default: 20000)
• --keep_significant_anchors_satc — if set there will be additional output file in SATC format with all significant anchors (default: False)
• --keep_top_target_entropy_anchors_satc — if set there will be additional output file in SATC format with top target entropy significant anchors (default: False)
• --keep_top_effect_size_bin_anchors_satc — if set there will be additional output file in SATC format with top effect size bin anchors (default: False)

Additional output configuration

• --dump_Cjs — output Cjs (default: False)
• --max_pval_opt_for_Cjs — dump only Cjs for anchors that have pval_opt <= max_pval_opt_for_Cjs (default: 0.1)
• --n_most_freq_targets — number of most frequent targets printed per each anchor (default: 10)
• --with_effect_size_cts — if set effect_size_cts will be computed (default: False)
• --with_pval_asymp_opt — if set pval_asymp_opt will be computed (default: False)
• --without_seqence_entropy — if set sequence entropy for anchor and most freq targets will not be computed (default: False)
• --sample_name_to_id — file name with mapping sample name <-> sample id (default: sample_name_to_id.mapping.txt)
• --dump_sample_anchor_target_count_txt — if set contingency tables will be generated in text format (default: False)
• --dump_sample_anchor_target_count_binary — if set contingency tables will be generated in binary (SATC) format, to convert to text format later satc_dump program may be used, it may take optionally mapping from id to sample_name (--sample_names param) (default: False)
• --supervised_test_samplesheet — if used script for finding/visualizing anchors with metadata-dependent variation will be run (forces --dump_sample_anchor_target_count_binary) (default: )
• --supervised_test_anchor_sample_fraction_cutoff — the cutoff for the minimum fraction of samples for each anchor (default: 0.4)
• --supervised_test_num_anchors — maximum number of anchors to be tested example (default: 20000)

Tuning statistics computation

• --opt_num_inits — the number of altMaximize random initializations (default: 10)
• --opt_num_iters — the maximum number of iterations in altMaximize (default: 50)
• --num_rand_cf — the number of random c and f used for pval_base (default: 50)
• --num_splits — the number of contingency table splits (default: 1)
• --opt_train_fraction — use this fraction to create training data from contingency table (default: 0.25)
• --without_alt_max — if set int alt max and related stats will not be computed (default: False)
• --without_sample_spectral_sum — if set sample spectral sum will not be computed (default: False)
• --Cjs_samplesheet — path to file with predefined Cjs for non-10X supervised mode (default: )

Technical and performance-related

• --bin_path — path to a directory where satc, satc_dump, satc_merge, sig_anch, kmc, kmc_tools, and other binaries are (if any not found there splash will check if installed and use installed) (default: bin)
• --tmp_dir — path to a directory where temporary files will be stored (default: let splash decide)

• --n_threads_stage_1 — number of threads for the first stage, too large value is not recomended because of intensive disk access here, but may be profitable if there is a lot of small size samples in the input (0 means auto adjustment) (default: 0)
• --n_threads_stage_1_internal — number of threads per each stage 1 thread (0 means auto adjustment) (default: 0)
• --n_threads_stage_1_internal_boost — multiply the value of n_threads_stage_1_internal by this (may increase performance but the total number of running threads may be high) (default: 1)
• --n_threads_stage_2 — number of threads for the second stage, high value is recommended if possible, single thread will process single bin (0 means auto adjustment) (default: 0)
• --n_bins — the data will be split in a number of bins that will be merged later (default: 128)
• --kmc_use_RAM_only_mode — True here may increase performance but also RAM-usage (default: False)
• --kmc_max_mem_GB — maximal amount of memory (in GB) KMC will try to not extend (default: 12)
• --dont_clean_up — if set then intermediate files will not be removed (default: False)
• --logs_dir — director where run logs of each thread will be stored (default: logs)

sc-SPLASH parameters (10x or Visium processing)

• --cbc_len — call barcode length (in case of 10X/Visium data) (default: 16)
• --umi_len — UMI length (in case of 10X/Visium data) (default: 12)
• --soft_cbc_umi_len_limit — allow additional symbols (beyond cbc_len + umi_len in _1.fastq 10X file UMI (default: 0)
• --cbc_filtering_thr — how to filter cbcs, if 0 do the same as umi tools, in the opposite case keep cbcs with freq >= <cbc_filtering_thr> (default: 0)
• --cell_type_samplesheet — path for mapping barcode to cell type, is used Helmert-based supervised mode is turned on (default: )
• --export_cbc_logs — use if need cbc log files (default: False)
• --predefined_cbc — path to file with predefined CBCs (default: )
• --export_filtered_input — use if need filtered FASTQ files (default: False)
• --allow_strange_cbc_umi_reads — use to prevent the application from crashing when the CBC+UMI read length is outside the acceptable range (either shorter than CBC+UMI or longer than CBC+UMI+soft_cbc_umi_len_limit) (default: False)