title: lorikeet call usage ---NAME

lorikeet call - Call variants within a given set of genomes and samples using local reassembly (version 0.8.2)

SYNOPSIS

lorikeet call [FLAGS]

DESCRIPTION

lorikeet call discovers variants within a given set of reads and genomes using a local reassembly algorithm based on the GATK HaplotypeCaller. Additionally, calculate strain diversity metrics like conANI, popANI, subpopANI, dN/dS, and the highly robust Hudson's Fst

This process can be undertaken in several ways, for instance by specifying BAM files or raw reads as input, using different mapping programs, thresholding read alignments.

FLAGS

-v, --verbose

: Print extra debugging information. [default: not set]

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-q, --quiet

: Unless there is an error, do not print log messages. [default: not set]

THREADING OPTIONS

-t, --threads INT

: Maximum number of threads used. [default: 10]

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-P, --parallel-genomes INT

: Number of genomes to run in parallel. Increases memory usage linearly. Thread usage qill not exceed the value provided by --threads [default 1]

INPUT REFERENCE OPTIONS

-r,-f, --reference,--genome-fasta-files PATH

: FASTA files of contigs e.g. concatenated genomes or metagenome assembly [required unless -d/--genome-fasta-directory is specified]

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-d, --genome-fasta-directory PATH

: Directory containing FASTA files of contigs e.g. genomes or metagenome assembly [required unless -r/--reference/-f/--genome-fasta-files is specified]

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-x, --genome-fasta-extension STR

: FASTA file extension in --genome-fasta-directory [default "fna"]

READ MAPPING PARAMETERS

-1 PATH ..

: Forward FASTA/Q file(s) for mapping. These may be gzipped or not.

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-2 PATH ..

: Reverse FASTA/Q file(s) for mapping. These may be gzipped or not.

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-c, --coupled PATH ..

: One or more pairs of forward and reverse possibly gzipped FASTA/Q files for mapping in order <sample1_R1.fq.gz> <sample1_R2.fq.gz> <sample2_R1.fq.gz> <sample2_R2.fq.gz> ..

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--interleaved PATH ..

: Interleaved FASTA/Q files(s) for mapping. These may be gzipped or not.

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--single PATH ..

: Unpaired FASTA/Q files(s) for mapping. These may be gzipped or not.

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--longreads PATH ..

: Longread FASTA/Q files(s) for mapping. These may be gzipped or not.

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-b, --bam-files PATH

: Path to BAM file(s). These must be reference sorted (e.g. with samtools sort) unless --sharded is specified, in which case they must be read name sorted (e.g. with samtools sort -n). When specified, no read mapping algorithm is undertaken.

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-l, --longread-bam-files PATH

: Path to longread BAM file(s). These must be reference sorted (e.g. with samtools sort) unless --sharded is specified, in which case they must be read name sorted (e.g. with samtools sort -n). When specified, no read mapping algorithm is undertaken.

SHARDING

--sharded

: If -b/--bam-files was used: Input BAM files are read-sorted alignments of a set of reads mapped to multiple reference contig sets. Choose the best hit for each read pair. Otherwise if mapping was carried out: Map reads to each reference, choosing the best hit for each pair. [default: not set]

MAPPING ALGORITHM OPTIONS

--mapper NAME

: Underlying mapping software used for short reads [default: minimap2-sr]. One of:

--longread-mapper NAME

: Underlying mapping software used for long reads [default: minimap2-ont]. One of:

--minimap2-params PARAMS

: Extra parameters to provide to minimap2, both indexing command (if used) and for mapping. Note that usage of this parameter has security implications if untrusted input is specified. '-a' is always specified to minimap2. [default: none]

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--minimap2-reference-is-index

: Treat reference as a minimap2 database, not as a FASTA file. [default: not set]

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--bwa-params PARAMS

: Extra parameters to provide to BWA or BWA-MEM2. Note that usage of this parameter has security implications if untrusted input is specified. [default: none]

ALIGNMENT THRESHOLDING

--min-read-aligned-length INT

: Exclude reads with smaller numbers of aligned bases. [default: 0]

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--min-read-percent-identity FLOAT

: Exclude reads by overall percent identity e.g. 95 for 95%. [default: 0]

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--min-read-aligned-percent FLOAT

: Exclude reads by percent aligned bases e.g. 95 means 95% of the read's bases must be aligned. [default: 0]

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--min-read-aligned-length-pair INT

: Exclude pairs with smaller numbers of aligned bases. Conflicts with --allow-improper-pairs. [default: 0]

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--min-read-percent-identity-pair FLOAT

: Exclude pairs by overall percent identity e.g. 95 for 95%. Conflicts with --allow-improper-pairs. [default: 0]

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--min-read-aligned-percent-pair FLOAT

: Exclude reads by percent aligned bases e.g. 95 means 95% of the read's bases must be aligned. Conflicts with --allow-improper-pairs. [default: 0]

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--allow-improper-pairs

: Require reads to be mapped as proper pairs. [default: not set]

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--exclude-supplementary

: Exclude supplementary alignments. [default: not set]

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--include-secondary

: Include secondary alignments. [default: not set]

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--contig-end-exclusion INT

: Exclude bases at the ends of reference sequences from calculation [default: 0]

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--trim-min FLOAT

: Remove this smallest fraction of positions when calculating trimmed_mean [default: 0.00]

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--trim-max FLOAT

: Maximum fraction for trimmed_mean calculations [default: 1.00]

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--split-bams

: Split the mapped read files up per reference. Useful if you think run time is being hampered by I/O. Most of the time this will not improve performance and instead just increase disk usage.

VARIANT CALLING OPTIONS (BASIC)

--profile STR

: Assembly profile to use for variant calling. Overrides --kmer-sizes, --min-prune-factor, --allow-non-unique-kmers-in-ref, and --recover-all-dangling-branches. Possible options include: 'fast', 'very-fast', 'sensitive', 'precise', 'super-sensitive' [default: not_set]

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-k, --kmer-sizes INT ..

: K-mer sizes used to generate DeBruijn Graphs. Multiple values at once are accepted and encouraged at a cost to increased runtime e.g. 17 21 25 [default: 21 33]

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--ploidy INT

: Sets the default ploidy for the analysis to N. [default: 2]

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--calculate-fst

: Calculate Fst values between samples and variants.

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--calculate-dnds

: Calculate coding regions and perform dN/dS calculations along them using called variants. *Microbial only*.

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-f, --features-vcf PATH

: The set of alleles to force-call regardless of evidence. Note: The sight containing these alleles has to be called as 'active' in order for them to appear in the final VCF. Addtionally, Provided file must be compressed using bgzip and indexed using bcftools index. If no index is present, and index will be attempted to be created. If the file is not properly compressed, Lorikeet will unfortunately SEGFAULT with no error message.

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--qual-by-depth-filter INT

: The minimum QD value for a variant to have for it to be included in the genotyping or ANI analyses. [default: 25]

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--qual-threshold INT

: The PHRED-scaled quality score threshold for use with ANI calculations. [default: 150]

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--depth-per-sample-filter INT

: Minimum depth of a variant in a sample for that sample to be included in ANI & Fst calculations for that variant. [default: 5]

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--min-long-read-size INT

: The minimum size for long reads to be used for analysis [default: 1500]

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--min-long-read-average-base-qual INT

: The minimum average base quality of a long read for it to be used for analysis [default: 20]

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-q, --min-base-quality INT

: Minimum base quality required to consider a base for calling. [default: 10]

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--min-mapq INT

: Minimum MAPQ score for reads to be considered during variant calling. [default: 20]

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--base-quality-score-threshold INT

: Base qualities below this threshold will be reduced to the minimum (6). [default: 18]

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--max-input-depth INT

: The maximum number of reads included within an assembly region across all samples. Larger numbers increase run time. If the depth of an assembly region exceeds this value, then the reads will be filtered by mean base quality. [default: 200000]

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--min-contig-size INT

: The minimum contig size to call variants on. Smaller contigs can often contain highly variable regions that mostly represent noise. Call variants on them can often be slow and not produce anything fruitful. [default: 0]

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--min-sv-qual INT

: Minimum structural variants quality returned by svim and used by lorikeet. Not PHRED-scaled quality, value determined by number of supporting reads. Consult svim documentation for details. [default: 3]

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--do-not-call-svs

: Opts not to use svim to call structural variants using provided longreads. If no longreads are provided this has no effect.

VARIANT CALLING OPTIONS (ADVANCED)

--phred-scaled-global-read-mismapping-rate INT

: The global assumed mismapping rate for reads. [default: 45]

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--pair-hmm-gap-continuation-penalty INT

: Flat gap continuation penalty for use in the Pair HMM. [default: 10]

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--pcr-indel-model STR

: The PCR indel model to use. [default: conservative]

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--heterozygosity FLOAT

: Heterozygosity value used to compute prior likelihoods for any locus. [default: 0.001]

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--heterozygosity-stdev FLOAT

: Standard deviation of heterozygosity for SNP and indel calling. [default: 0.01]

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--indel-heterozygosity FLOAT

: Heterozygosity for indel calling. [default: 0.000125]

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-C, --standard-min-confidence-threshold-for-calling FLOAT

: The minimum phred-scaled confidence threshold at which variants should be called. [default: 25.0]

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--use-posteriors-to-calculate-qual

: if available, use the genotype posterior probabilities to calculate the site QUAL.

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--annotate-with-num-discovered-alleles

: If provided, we will annotate records with the number of alternate alleles that were discovered (but not necessarily genotyped) at a given site.

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--active-probability-threshold FLOAT

: Minimum probability for a locus to be considered active. [default: 0.002]

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--min-assembly-region-size INT

: Minimum size of an assembly region. [default: 50]

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--max-assembly-region-size INT

: Maximum size of an assembly region. [default: 300]

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--assembly-region-padding INT

: Number of additional bases of context to include around each assembly region. [default: 100]

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--dont-increase-kmer-sizes-for-cycles

: Disable iterating over kmer sizes when graph cycles are detected.

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--allow-non-unique-kmers-in-ref

: Allow graphs that have non-unique kmers in the reference. Can increase runtime and sensitivity in repeat regions.

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--do-not-run-physical-phasing

: Disable physical phasing.

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--recover-all-dangling-branches

: Recover all dangling branches.

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--min-dangling-branch-length INT

: Minimum length of a dangling branch to attempt recovery. [default: 1]

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--min-prune-factor INT

: Minimum read support to not prune paths in the graph. Lower values like 1 or 0 will drastically increase runtime, but recover more variants. For faster performance, set this to 2 or 3 at a cost to recall. [default: 1]

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--use-adaptive-pruning

: Use more advanced pruning algorithm to prune paths in graph. Better suited when performing variant calling on when depth along a genome is variable e.g. RNA and exome data.

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--num-pruning-samples INT

: Number of samples that must pass the min_pruning threshold [default: 1]

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--graph-output PATH

: Write debug assembly graph information to this file.

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--dont-use-soft-clipped-bases

: Do not analyse soft clipped bases in the reads.

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--initial-error-rate-for-pruning FLOAT

: Initial base error rate estimate for adaptive pruning. [default: 0.001]

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--pruning-log-odds-threshold FLOAT

: Likelihood ratio threshold for adaptive pruning algorithm. This value will be converted to log odds value. [default: 1.0]

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--max-unpruned-variants INT

: Maximum number of variants in graph the adaptive pruner will allow. [default: 100]

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--max-prob-propagation-distance INT

: Upper limit on how many bases away probability mass can be moved around when calculating the boundaries between active and inactive assembly regions. [default: 50]

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--max-mnp-distance INT

: Two or more phased substitutions separated by this distance or less are merged into MNPs. [default: 0]

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--disable-optimizations

: Don't skip calculations in ActiveRegions with no variants

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--disable-avx

: Disable the use of the GKL-rs AVX acceleration components for PairHMM and Smith-Waterman calculations.

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--limiting-interval STR

: Mainly used for debugging purposes. Only call variants within this given span on all contigs. E.g. providing '1000-2000' would only call variants between the 1000 and 2000 bp span on each provided contig.

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--force

: Forcefully overwrite previous runs.

OUTPUT OPTIONS

-o, --output-directory DIRECTORY

: Output directory. Folder will contain subfolders for each input genome

[default: ./]

--bam-file-cache-directory DIRECTORY

: Output BAM files generated during alignment to this directory. The directory may or may not exist. Note that BAM files in this directory contain all mappings, including those that later are excluded by alignment thresholding (e.g. --min-read-percent-identity) or genome-wise thresholding (e.g. --min-covered-fraction). [default: not used]

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--keep-unmapped

: Include unmapped reads from cached BAM files. [default: not set]

FREQUENTLY ASKED QUESTIONS (FAQ)

Can the temporary directory used be changed? Lorikeet makes use of the system temporary directory (often /tmp) to store intermediate files. This can cause problems if the amount of storage available there is small or used by many programs. To fix, set the TMPDIR environment variable e.g. to set it to use the current directory: TMPDIR=. lorikeet call <etc>

EXIT STATUS

: Successful program execution.

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: Unsuccessful program execution.

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101

: The program panicked.

EXAMPLES

Map paired reads to a reference call variants and calculate Fst

: $ lorikeet call --coupled read1.fastq.gz read2.fastq.gz --reference assembly.fna --threads 10 --kmer-sizes 10 25 51 --calculate-fst

Call variants from read mappings compared to reference from a sorted BAM file and plots the results

: $ lorikeet call --bam-files my.bam --longread-bam-files my-longread.bam --genome-fasta-directory genomes/ -x fna --bam-file-cache-directory saved_bam_files --output-directory lorikeet_out/ --threads 10

AUTHOR

Rhys J. P. Newell, Centre for Microbiome Research, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology <rhys.newell94 near gmail.com>