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skovaka / uncalled4

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Uncalled4

Utility for Nanopore Current Alignment to Large Expanses of DNA (version 4)

A toolkit for nanopore signal alignment, analysis, and visualization

Performs accurate basecaller-guided nanopore signal alignment, similar to nanopolish eventalign or tombo resquiggle, to map nanopore signal segments to reference nucleotides. Also supports conversion of any signal alignments to an efficient BAM format, allowing for interactive visualizations, modification detection, and other signal analyses.

For real-time targeted sequencing via rapid signal mapping, see UNCALLED

Read our pre-print on BioRxiv!

Uncalled4 improves nanopore DNA and RNA modification detection via fast and accurate signal alignment \ S. Kovaka, P.W. Hook, K.M. Jenike, V. Shivakumar, L.B. Morina, R. Razaghi, W. Timp, M.C. Schatz \ BioRxiv (2024)

Table of Contents

Installation

pip install uncalled4

OR

git clone https://github.com/skovaka/uncalled4.git
cd uncalled4
pip install .

Requires python >= 3.8 and GCC>=4.9.2

Uncalled4 is currently only tested Linux, primarily on Ubuntu 18.04 and 20.04

Overview

The example/ directory contains scripts to download example data and test each command:

cd uncalled4/example
./download.sh
./run.sh

Uncalled4 signal alignment is guided by BAM alignments output by the basecaller with "move" tags (Dorado --emit-moves --emit-sam or Guppy --moves_out), which include mv:, ts:, and related BAM tags. This BAM file should contain reference-aligned reads (Dorado --reference or guppy --align_ref), unmapped will be ignored. To (re)align reads while preserving these tags, you can use samtools fastq -T "mv,ts,pi,sp,ns" <file>.bam > file.fastq (v1.16 or newer) to convert the tags to FASTQ format, then align using minimap2 -y ... to propagate the tags to the SAM file. Guppy only includes BAM tag on primary alignments by default. See scripts/bamprep.py to copy tags to supplemental and secondary aligments.

Uncalled4 primarily stores signal alignments in BAM alignment tags, including per-reference signal coordinates and current summary statistics required for most signal analyses. A sorted and indexed (via samtools index) Uncalled4 BAM file is required for most visualization and analysis commands. Nanopolish, f5c, or Tombo alignments can be converted to Uncalled4 format via the convert command.

Signal alignment requires a pore model to map k-mers to expected current. Uncalled4 will attempt to automatically detect the appropriate pore model from the input data, but may require you to specify a preset pore model or custom pore model. This can be specified using the --pore-model flag or --flowcell and --kit flags. uncalled4 train trains new pore models, either starting from a initialization pore model, or from scratch using basecaller moves.

Preliminary support for RNA004 alignment is implemented on the dev branch. Signal alignment quality appears to be better than RNA002, but the exact implementation may change before migrating to the main branch.

align

Perform DTW alignment guided by basecalled alignments

uncalled4 align [-h] [--ref REF | --self] [--reads READS [READS ...]] --bam-in [BAM_IN] [-p PROCESSES] [--flowcell FLOWCELL]
                [--kit KIT] [--basecaller-profile BASECALLER_PROFILE] [--rna] [--ordered-out] [-f] [--kmer-shift KMER_SHIFT]
                [--bam-chunksize BAM_CHUNKSIZE] [--out-name OUT_NAME] [-r] [-l READ_FILTER] [-x READ_INDEX] [-n MAX_READS]
                [--count-events] [--del-max DEL_MAX] [--ins-max INS_MAX] [--unmask-splice] [--method METHOD] 
                [-c {abs_diff,z_score,norm_pdf}] [-b BAND_WIDTH] [-s BAND_SHIFT] [--mvcmp-mask MVCMP_MASK]
                [--max-norm-dist MAX_NORM_DIST] [--max-sd MAX_SD] [--min-aln-length MIN_ALN_LENGTH] [-N {ref_mom,model_mom}]
                [--zero-ts] [-C CONFIG] [-o [BAM_OUT] | --tsv-out [TSV_OUT] | --eventalign-out [EVENTALIGN_OUT]] [-m PORE_MODEL]
                [--bam-f5c] [--tsv-cols TSV_COLS] [--tsv-na [TSV_NA]] [--tsv-noref] [--eventalign-flags EVENTALIGN_FLAGS]       
                [--norm-iterations NORM_ITERATIONS] [--mask-skips [MASK_SKIPS]] [--skip-cost SKIP_COST] [--stay-cost STAY_COST]
                [--move-cost MOVE_COST]                                                                                         

required arguments:
  --ref [fasta] | --self     Reference to align to, or perform signal-to-read self-alignment
  --reads [path]             Paths to fast5, slow5, or pod5 files, or to directories containing those files (optionally recursive)
  --bam-in [bam_in]          BAM input file (or "-"/no argument for stdin) (default: None)

output arguments (one required):
  -o, --bam-out     BAM output file
  --tsv-out         TSV output file (or "-"/no argument for stdout) 
  --eventalign-out  Eventalign (nanopolish) output file (or "-"/no argument for stdout)

selected optional arguments:
  -p, --processess      Number of parallel processes (default: 1)
  --flowcell FLOWCELL   Flowcell used for sequencing (e.g. FLO-MIN106)
  --kit KIT             Kit used for sequencing (e.g. SQK-LSK109)
  -m, --pore-model       Custom pore model
  --rna                 Required for custom RNA pore models
  --tsv-cols TSV_COLS   TSV file output alignment layers (comma-separated, can also include "read_id" (default: dtw)
  --tsv-na [TSV_NA]     Missing value representation for TSV output (default: *)
  --tsv-noref           Will NOT output reference coordinates to TSV if True (default: False)
  --eventalign-flags    Eventalign optional flags (comma-separated list of ""print-read-names", "signal-index", "samples")
  -h, --help            Print full command line usage

--bam-in must be a BAM file produced by Dorado using --emit-moves --emit-sam flags or the Guppy --moves_out flag.

The --self option is an alternative to --ref, and performs signal-to-read alignment to the basecalled read, which must be defined in the SEQ field in the input BAM file.

convert

Convert between signal alignment file formats

uncalled4 convert [-h] [--bam-in BAM_IN | --eventalign-in [EVENTALIGN_IN] | --tombo-in TOMBO_IN]   
                  [--eventalign-out [EVENTALIGN_OUT] | --tsv-out [TSV_OUT] | 
                  --m6anet-out [M6ANET_OUT]] [--tsv-cols TSV_COLS] 
                  [--eventalign-flags EVENTALIGN_FLAGS]
                  [--mask-skips [MASK_SKIPS]] [--ref FASTA] [--reads READS [READS ...]]          
                  [-l READ_FILTER] [-x READ_INDEX] [-r] [--rna] [-R REF_BOUNDS] [-f] [-a]

Generally only one --*-in and one --*-out option should be specified, with the exception of --bam-out where a template bam file should be specified via --bam-in.

nanopolish or f5c eventalign should be run with the --signal-index and --scale-events options, and can be converted with uncalled4 convert --eventalign-in <eventalign.txt> --bam-in <mm2.bam>, where <mm2.bam> is the exact BAM file used to guide the eventalign command.

--m6anet-out efficently implements m6anet dataprep for sorted Uncalled4 BAM files. This should be used with an m6Anet model trained on Uncalled4 alignments

train

Iteratively train a new k-mer pore model

Accepts most of the same paramters as uncalled4 align, in addition to number of iterations. First iteration must use some starting pore model, while subsequent iterations use the pore model from the previous iteration.

uncalled4 train [-h] [-i TRAIN_ITERATIONS] [-m INIT_MODEL] [--init-mode INIT_MODE] [--moves-avg MOVES_AVG] [-k KMER_LEN]
                [--kmer-samples KMER_SAMPLES] [--buffer-size BUFFER_SIZE] [-d MAX_MOVES_DIST] [--train-mean] [--out-dir OUT_DIR] [-a]
                [--skip-dtw] [--mask-skips [MASK_SKIPS]] [--norm-iterations NORM_ITERATIONS] [--skip-cost SKIP_COST]
                [--stay-cost STAY_COST] [--move-cost MOVE_COST] [--ref REF | --self] [--reads READS [READS ...]] --bam-in [BAM_IN]
                [-p PROCESSES] [--flowcell FLOWCELL] [--kit KIT] [--basecaller-profile BASECALLER_PROFILE] [--rna] [--ordered-out]
                [-f] [--kmer-shift KMER_SHIFT] [--bam-chunksize BAM_CHUNKSIZE] [--out-name OUT_NAME] [-r] [-l READ_FILTER]
                [-x READ_INDEX] [-n MAX_READS] [--count-events] [--del-max DEL_MAX] [--ins-max INS_MAX] [--unmask-splice]
                [--method METHOD] [-c {abs_diff,z_score,norm_pdf}] [-b BAND_WIDTH] [-s BAND_SHIFT] [--mvcmp-mask MVCMP_MASK]
                [--max-norm-dist MAX_NORM_DIST] [--max-sd MAX_SD] [--min-aln-length MIN_ALN_LENGTH] [-N {ref_mom,model_mom}]
                [--zero-ts] [-C CONFIG]

Visualization

All visualizations are generated using Plotly.

dotplot

Plot signal-to-reference alignment dotplots

uncalled4 dotplot [-h] [--bam-in BAM_IN [BAM_IN ...]] [-o OUT_PREFIX] [--ref REF] [--names NAMES] [--reads READS [READS ...]]
                  [-x READ_INDEX] [-r] [--rna] [-f {svg,png,pdf}] [-R REGION] [-l READ_FILTER] [-L LAYERS] [-p PORE_MODEL]
                  [--multi-background] [--no-model] [--svg] [-C CONFIG]

options:
  -h, --help            show this help message and exit
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  -o OUT_PREFIX, --out-prefix OUT_PREFIX
                        If included will output images with specified prefix, otherwise will display interactive plot. (default: None)
  --ref REF             Reference FASTA file, must match --bam-in reference (default: None)
  --names NAMES         Names of tracks to read from input(s) (default: None)
  --reads READS [READS ...]
                        Paths to FAST5, SLOW5, or POD5 files, or to directories containing those files (optionally recursive) (default:
                        None)
  -x READ_INDEX, --read-index READ_INDEX
                        File containing a mapping of read IDs to filenames (default: None)
  -r, --recursive       Recursively search 'paths' for FAST5, SLOW5, or POD5 files (default: False)
  --rna                 Should be set for direct RNA data (default: None)
  -f {svg,png,pdf}, --out-format {svg,png,pdf}
                        Image output format. Only has an effect with -o option. (default: svg)
  -R REGION, --region REGION
                        Only load reads which overlap these coordinates (default: None)
  -l READ_FILTER, --read-filter READ_FILTER
                        List of read IDs to load, or file containing one read ID per line (default: None)
  -L LAYERS, --layers LAYERS
  -p PORE_MODEL, --pore-model PORE_MODEL
                        Model preset name or TSV filename (default: None)
  --multi-background    Will plot multiple stacked background colors for multiple tracks if True (default: False)
  --no-model            Will not plot the expected reference signal if True (default: False)
  --svg                 Make SVG-friendly figures (default: False)
  -C CONFIG, --config CONFIG
                        Configuration file in TOML format (default: None)

trackplot

Plot alignment tracks and per-reference statistics

Trackplots are defined by a series of panels displaying different layers. A mat panel display a heatmap of layer values for each ref/read coordinate on each track. A box panel displays boxplots of layer summary statistics for each track. line and scatter panels display refstats summary statistics, specified by <layer>.<statistic> (e.g. current.mean, model_diff.median).

uncalled4 trackplot [-h] -R REGION --bam-in BAM_IN [BAM_IN ...] [--ref REF] [--read-paths READ_PATHS [READ_PATHS ...]]
                    [-x READ_INDEX] [-r] [--rna] [--pore-model PORE_MODEL] [--svg] [-f] [-l READ_FILTER]
                    [-H PANEL_HEIGHTS [PANEL_HEIGHTS ...]] [--shared-refs-only] [--shared-reads-only] [--share-reads] [--hover-read]
                    [-o OUTFILE] [-C CONFIG] [--bases] [--mat LAYER] [--box LAYER] [--line LAYER.STAT] [--scatter LAYER.STAT]

options:
  -h, --help            show this help message and exit
  -R REGION, --region REGION
                        Only load reads which overlap these coordinates (default: None)
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  --ref REF             Reference FASTA file, must match --bam-in reference (default: None)
  --read-paths READ_PATHS [READ_PATHS ...]
                        Paths to FAST5, SLOW5, or POD5 files, or to directories containing those files (optionally recursive) (default:
                        None)
  -x READ_INDEX, --read-index READ_INDEX
                        File containing a mapping of read IDs to filenames (default: None)
  -r, --recursive       Recursively search 'paths' for FAST5, SLOW5, or POD5 files (default: False)
  --rna                 Should be set for direct RNA data (default: None)
  --pore-model PORE_MODEL
                        Model preset name or TSV filename (default: )
  --svg                 Make SVG-friendly figures (default: False)
  -f, --full-overlap    If true will only include reads which fully cover reference bounds (default: False)
  -l READ_FILTER, --read-filter READ_FILTER
                        List of read IDs to load, or file containing one read ID per line (default: None)
  -H PANEL_HEIGHTS [PANEL_HEIGHTS ...], --panel-heights PANEL_HEIGHTS [PANEL_HEIGHTS ...]
                        Relative height of each panel (default: None)
  --shared-refs-only    If true will only contain reference positions where all tracks have sufficient coverage (see min_coverage) (default:
                        False)
  --shared-reads-only   If true will only contain reads shared between all tracks (default: False)
  --share-reads         If True will only display reads shared by all alignment tracks with shared y-axis (default: False)
  --hover-read          If True will display read_id in mat hover (default: False)
  -o OUTFILE, --outfile OUTFILE
                        Output file (default: None)
  -C CONFIG, --config CONFIG
                        Configuration file in TOML format (default: None)
  --bases               Display a ref-by-read matrix of specified alignment layer (default: None)
  --mat LAYER           Display a ref-by-read matrix of specified alignment layer (default: None)
  --box LAYER           Display a boxplot of specified layer (default: None)
  --line LAYER.STAT     Display a line plot of specifed layer summary statistic (default: None)
  --scatter LAYER.STAT  Display a line plot of specifed layer summary statistic (default: None)

browser

Interactive signal alignment genome browser

Integrates trackplot and dotplot for interactive alignment browsing

uncalled4 browser [-h] -R REGION [--bam-in BAM_IN [BAM_IN ...]] [--shared-reads-only] [--reads READS [READS ...]] [--ref REF]
                  [-x READ_INDEX] [-r] [--rna] [-l READ_FILTER] [-f] [--pore-model PORE_MODEL] [--names NAMES] [-p PORT] [-o OUTFILE]
                  [-C CONFIG]

options:
  -h, --help            show this help message and exit
  -R REGION, --region REGION
                        Reference coordinates to visualize (chr:start-end) (default: None)
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  --shared-reads-only   If true will only contain reads shared between all tracks (default: False)
  --reads READS [READS ...]
                        Paths to FAST5, SLOW5, or POD5 files, or to directories containing those files (optionally recursive) (default:
                        None)
  --ref REF             Reference FASTA file, must match --bam-in reference (default: None)
  -x READ_INDEX, --read-index READ_INDEX
                        File containing a mapping of read IDs to filenames (default: None)
  -r, --recursive       Recursively search 'paths' for FAST5, SLOW5, or POD5 files (default: False)
  --rna                 Should be set for direct RNA data (default: None)
  -l READ_FILTER, --read_filter READ_FILTER
                        Only load reads which overlap these coordinates (default: None)
  -f, --full-overlap    If true will only include reads which fully cover reference bounds (default: False)
  --pore-model PORE_MODEL
                        Model preset name or TSV filename (default: )
  --names NAMES         Names of tracks to read from input(s) (default: None)
  -p PORT, --port PORT  Browser port (default: 8000)
  -o OUTFILE, --outfile OUTFILE
                        Output file (default: None)
  -C CONFIG, --config CONFIG
                        Configuration file in TOML format (default: None)

Analysis

These functions compute statistics over reference and read coordinates. refstats computes summary and comparison statistics (e.g. Kolmogorov-Smirnov test) over reference coordinates, while layerstats maintains the read-by-reference dimensions of the DTW alignments.

(Coming soon: readstats to compute read-level statistics)

refstats

Calculate per-reference-coordinate statistics

uncalled4 refstats [-h] [--bam-in BAM_IN [BAM_IN ...]] [--layers LAYERS [LAYERS ...]] [--stats REFSTATS [REFSTATS ...]] 
                   [-t TRACKS] [-R REGION] [--min-coverage MIN_COVERAGE] [--bed-filter BED_FILTER] [--ref-chunksize REF_CHUNKSIZE] 
                   [--aln-chunksize ALN_CHUNKSIZE] [-c] [--ref REF] [-m PORE_MODEL] [-p PROCESSES] [-o OUTFILE]

required arguments:
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  --layers LAYERS [LAYERS ...]
                        Comma-separated list of layers over which to compute summary statistics (default: [])
  --stats REFSTATS [REFSTATS ...]
                        Comma-separated list of summary statistics to compute. Some statisitcs (ks) can only be used if exactly two tracks
                        are provided {q5,kurt,q75,KS,q95,max,var,skew,median,stdv,mean,q25,min} (default: None)

options:
  -h, --help            show this help message and exit
  -t TRACKS, --tracks TRACKS
                        Names of tracks to read from input(s) (default: None)
  -R REGION, --region REGION
                        Only load reads which overlap these coordinates (default: None)
  --min-coverage MIN_COVERAGE
                        Reference positions with less than this coverage will be excluded from each track (or all tracks if shared_refs_only
                        is true) (default: 1)
  --bed-filter BED_FILTER
                        Only parse regions in BED file (default: None)
  --ref-chunksize REF_CHUNKSIZE
                        Number of reference coordinates to query for iteration (default: 10000)
  --aln-chunksize ALN_CHUNKSIZE
                        Number of alignments to query for iteration (default: 500)
  -c, --cov             Output track coverage for each reference position (default: False)
  --ref REF             Reference FASTA file, must match --bam-in reference (default: None)
  -m PORE_MODEL, --pore-model PORE_MODEL
                        Model preset name or TSV filename (default: None)
  -p PROCESSES, --processes PROCESSES
                        Number of parallel processes (default: 1)
  -o OUTFILE, --outfile OUTFILE

readstats

Compute per-read summary statistics

uncalled4 readstats [-h] [--bam-in BAM_IN [BAM_IN ...]] [--layers LAYERS [LAYERS ...]] [--stats STATS [STATS ...]] [-R REGION]
                    [-s SUMMARY_STATS] [-C CONFIG]

options:
  -h, --help            show this help message and exit
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  --layers LAYERS [LAYERS ...]
                        Which layers to compute statistics (default: None)
  --stats STATS [STATS ...]
                        Summary statistics to compute (any builtin numpy function, e.g. mean, std, etc) (default: None)
  -R REGION, --region REGION
                        Only load reads which overlap these coordinates (default: None)
  -s SUMMARY_STATS, --summary-stats SUMMARY_STATS
                        Summary statistics to compute for "model_diff" command. (default: ['mean'])
  -C CONFIG, --config CONFIG
                        Configuration file in TOML format (default: None)

compare

Compute distance between alignments of the same reads

uncalled4 compare [-h] [--bam-in BAM_IN [BAM_IN ...]] [-t TRACKS] [-l READ_FILTER] [-R REGION] [-m] [--tsv-cols TSV_COLS]
                  [--tsv-na [TSV_NA]] [--tsv-noref] [--tsv-out [TSV_OUT]] [-C CONFIG]

options:
  -h, --help            show this help message and exit
  --bam-in BAM_IN [BAM_IN ...]
                        BAM input file (default: None)
  -t TRACKS, --tracks TRACKS
                        Names of tracks to read from input(s) (default: None)
  -l READ_FILTER, --read-filter READ_FILTER
                        Only load reads which overlap these coordinates (default: None)
  -R REGION, --region REGION
                        Only load reads which overlap these coordinates (default: None)
  -m, --moves           Compare against basecalled alignment. If two tracks input will look for "moves" group in second track, otherwise
                        will look in the first track. (default: False)
  --tsv-cols TSV_COLS   TSV file output alignment layers (comma-separated, can also include "read_id" (default: dtwcmp,mvcmp)
  --tsv-na [TSV_NA]     Missing value representation for TSV output (default: *)
  --tsv-noref           Will NOT output reference coordinates to TSV if True (default: False)
  --tsv-out [TSV_OUT], -o [TSV_OUT]
                        TSV output file (or "-"/no argument for stdout) (default: -)
  -C CONFIG, --config CONFIG
                        Configuration file in TOML format (default: None)

Pore Models

Uncalled4 pore models map k-mers to their expected current characteristics for a specific sequencing chemistry, minimally defining the expected mean current (current.mean) for each k-mer. Pore models trained by Uncalled4 include means and standard deviations for per-kmer current means, current standard deviations, and dwell time measured in raw sample length: current.mean/current.stdv, current_sd.mean/current_sd.stdv, length.mean/length.stdv. In addition to per-kmer statistics, each model has a defined k-mer shift used to define the central base that has the most effect on the current, the picoamp mean and standard deviation (pa_mean and pa_stdv) that can be used to scale the normalized current values to picoamps, and other model-specifc parameters like sample_rate and bases_per_sec. A reverse parameter is also included, which is set to True for RNA to indicated reversed sequencing direction.

Four pore model presets are provided by Uncalled4: dna_r10.4.1_400bps_9mer, dna_r9.4.1_400bps_6mer, rna_r9.4.1_70bps_5mer, and rna004_130bps_9mer. These are stored efficently in binary NumPy "npz" files, and can be converted to TSV format using the provided model2tsv.py script.

Custom pore models can be provided in TSV format via the --pore-model command line argument, which should minimally include columns named kmer and current.mean. Column names for current levels are also aliased to support Nanopolish and other similar models, so current.mean can be level_mean or mean, current_sd.mean can be sd_mean or stdv, etc. K-mer offsets can also be defined for custom pore models using the --kmer-shift option. All pore models are automatically normalized such that the mean and standard deviation of current.mean is 0 and 1 respecively, which is required for BAM encoding, and the resulting BAM file will store scaling factors to convert to the original input values in pa_mean and pa_stdv.

Uncalled4 will attempt to automatically detect the sequencing chemistry based on POD5/FAST5/BLOW5 metadata, which is required even with custom pore models to determine the appropriate offset to use for basecaller moves metadata. If this cannot be automatically detected, the --basecaller-profile must also be provided, which is defined similar to the pore model presets but without a defined k-mer length: either dna_r10.4.1_400bps, dna_r10.4.1_260bps, dna_r9.4.1_400bps, rna_r9.4.1_70bps, or rna004_130bps. If you are using a sequenicng chemistry which does not have an appropirate preset, please submit an issue and we can implement one.

Alignment Layers

Uncalled4 stores signal alignments as a set of layers associated with read and reference coordinates. Each layer is derived from the read signal (e.g. current), the reference sequence (e.g. kmer), or other layers (e.g. model_diff). Layers are organized into layer groups: dtw for signal alignments, bcaln for projected basecalled alignments, and cmp for alignment comparisons. Several subcommands detailed above take layer names as input, which should generaly be in the form <group>.<layer>. Below is a table of currently available layers:

Group Layer Description
dtw current Normalized mean read signal current
dtw current_sd Normalized read signal current standard deviation
dtw start Read signal sample start index
dtw length Read signal sample length
dtw dwell Signal dwell time (ms/nt, proportional to length)
dtw model_diff Difference between predicted (via a pore model) and observed current
dtw abs_diff Absolute value of dtw.model_diff
dtw kmer Binarized reference k-mer
dtw events Number of raw signal events aligned ("stays" > 1, "skips" < 1)
dtw base Binarized reference base
moves start Estimated read signal sample start index
moves length Estimated read signal sample length
moves indel Number of inserted (>0) or deleted (<0) nucleotides
mvcmp mean_ref_dist Mean reference distance between signal alignment and ref-moves
mvcmp jaccard Raw sample jaccard distances between signal alignment and ref-moves
dtwcmp mean_ref_dist Mean reference distance between two alignments (must first run layerstats compare)
dtwcmp jaccard Raw sample jaccard distances between two alignments (must first run layerstats compare)

All tracks must be written to the same database for multi-track visualization and analysis (e.g. comparing alignments, calculating KS statistics). You can merge multiple databases into a single file using uncalled db merge

Release Notes