Usage
Michael Hall edited this page May 10, 2021
·
5 revisions
- Synopsis
- Population Reference Graphs
- Build index
- Map reads to index
- Compare reads from several samples
- Discover novel variants
$ pandora --help
Pandora: Pan-genome inference and genotyping with long noisy or short accurate reads.
Usage: pandora [OPTIONS] SUBCOMMAND
Options:
-h,--help Print this help message and exit
-V,--version
Subcommands:
index Index population reference graph (PRG) sequences.
map Quasi-map reads to an indexed PRG, infer the sequence of present loci in the sample, and optionally genotype variants.
compare Quasi-map reads from multiple samples to an indexed PRG, infer the sequence of present loci in each sample, and call variants between the samples.
discover Quasi-map reads to an indexed PRG, infer the sequence of present loci in the sample and discover novel variants.
walk Outputs a path through the nodes in a PRG corresponding to the either an input sequence (if it exists) or the top/bottom path
seq2path For each sequence, return the path through the PRG
get_vcf_ref Outputs a fasta suitable for use as the VCF reference using input sequences
random Outputs a fasta of random paths through the PRGs
merge_index Allows multiple indices to be merged (no compatibility check)
Pandora assumes you have already constructed a fasta-like file of graphs, one entry for each gene/ genome region of interest. If you haven't, you will need a multiple sequence alignment for each graph. Precompiled collections of MSA representing othologous gene clusters for a number of species can be downloaded from here and converted to graphs using the pipeline from here.
Takes a fasta-like file of PanRG sequences and constructs an index, and
a directory of gfa files to be used by pandora map or pandora compare. These are output in the same directory as the PanRG file.
$ pandora index --help
Index population reference graph (PRG) sequences.
Usage: pandora index [OPTIONS] <PRG>
Positionals:
<PRG> FILE [required] PRG to index (in fasta format)
Options:
-h,--help Print this help message and exit
-w INT Window size for (w,k)-minimizers (must be <=k) [default: 14]
-k INT K-mer size for (w,k)-minimizers [default: 15]
-t,--threads INT Maximum number of threads to use [default: 1]
-o,--outfile FILE Filename for the index [default: <PRG>.kXX.wXX.idx]
-v Verbosity of logging. Repeat for increased verbosity
The index stores (w,k)-minimizers for each PanRG path found. These parameters can be specified, but default to w=14, k=15.
This takes a fasta/q of Nanopore or Illumina reads and compares to the index. It infers which of the PanRG genes/elements is present, and for those that are present it outputs the inferred sequence and a genotyped VCF.
$ pandora map --help
Quasi-map reads to an indexed PRG, infer the sequence of present loci in the sample, and optionally genotype variants.
Usage: ./pandora map [OPTIONS] <TARGET> <QUERY>
Positionals:
<TARGET> FILE [required] An indexed PRG file (in fasta format)
<QUERY> FILE [required] Fast{a,q} file containing reads to quasi-map
Options:
-h,--help Print this help message and exit
-v Verbosity of logging. Repeat for increased verbosity
Indexing:
-w INT Window size for (w,k)-minimizers (must be <=k) [default: 14]
-k INT K-mer size for (w,k)-minimizers [default: 15]
Input/Output:
-o,--outdir DIR Directory to write output files to [default: pandora]
-t,--threads INT Maximum number of threads to use [default: 1]
--vcf-refs FILE Fasta file with a reference sequence to use for each loci. The sequence MUST have a perfect match in <TARGET> and the same name
--kg Save kmer graphs with forward and reverse coverage annotations for found loci
--loci-vcf Save a VCF file for each found loci
-C,--comparison-paths Save a fasta file for a random selection of paths through loci
-M,--mapped-reads Save a fasta file for each loci containing read parts which overlapped it
Parameter Estimation:
-e,--error-rate FLOAT Estimated error rate for reads [default: 0.11]
-g,--genome-size STR/INT Estimated length of the genome - used for coverage estimation. Can pass string such as 4.4m, 100k etc. [default: 5000000]
--bin Use binomial model for kmer coverages [default: negative binomial]
Mapping:
-m,--max-diff INT Maximum distance (bp) between consecutive hits within a cluster [default: 250]
-c,--min-cluster-size INT Minimum size of a cluster of hits between a read and a loci to consider the loci present [default: 10]
Preset:
-I,--illumina Reads are from Illumina. Alters error rate used and adjusts for shorter reads
Filtering:
--clean Add a step to clean and detangle the pangraph
--max-covg INT Maximum coverage of reads to accept [default: 300]
Consensus/Variant Calling:
--genotype Add extra step to carefully genotype sites.
--snps When genotyping, only include SNP sites
--kmer-avg INT Maximum number of kmers to average over when selecting the maximum likelihood path [default: 100]
Genotyping:
--local Needs: --genotype (Intended for developers) Use coverage-oriented (local) genotyping instead of the default ML path-oriented (global) approach.
-a INT Hard threshold for the minimum allele coverage allowed when genotyping [default: 0]
-s INT The minimum required total coverage for a site when genotyping [default: 0]
-D INT Minimum difference in coverage on a site required between the first and second maximum likelihood path [default: 0]
-F INT Minimum allele coverage, as a fraction of the expected coverage, allowed when genotyping [default: 0]
-E,--gt-error-rate FLOAT When genotyping, assume that coverage on alternative alleles arises as a result of an error process with rate -E. [default: 0.01]
-G,--gt-conf INT Minimum genotype confidence (GT_CONF) required to make a call [default: 1]
This takes Nanopore or Illumina read fasta/q for a number of samples, mapping each to the index. It infers which of the PanRG genes/elements is present in each sample, and outputs a presence/absence pangenome matrix, the inferred sequences for each sample and a genotyped multisample pangenome VCF.
$ pandora compare --help
Quasi-map reads from multiple samples to an indexed PRG, infer the sequence of present loci in each sample, and call variants between the samples.
Usage: ./pandora compare [OPTIONS] <TARGET> <QUERY_IDX>
Positionals:
<TARGET> FILE [required] An indexed PRG file (in fasta format)
<QUERY_IDX> FILE [required] A tab-delimited file where each line is a sample identifier followed by the path to the fast{a,q} of reads for that sample
Options:
-h,--help Print this help message and exit
-v Verbosity of logging. Repeat for increased verbosity
Indexing:
-w INT Window size for (w,k)-minimizers (must be <=k) [default: 14]
-k INT K-mer size for (w,k)-minimizers [default: 15]
Input/Output:
-o,--outdir DIR Directory to write output files to [default: pandora]
-t,--threads INT Maximum number of threads to use [default: 1]
--vcf-refs FILE Fasta file with a reference sequence to use for each loci. The sequence MUST have a perfect match in <TARGET> and the same name
--loci-vcf Save a VCF file for each found loci
Parameter Estimation:
-e,--error-rate FLOAT Estimated error rate for reads [default: 0.11]
-g,--genome-size STR/INT Estimated length of the genome - used for coverage estimation. Can pass string such as 4.4m, 100k etc. [default: 5000000]
--bin Use binomial model for kmer coverages [default: negative binomial]
Mapping:
-m,--max-diff INT Maximum distance (bp) between consecutive hits within a cluster [default: 250]
-c,--min-cluster-size INT Minimum size of a cluster of hits between a read and a loci to consider the loci present [default: 10]
Preset:
-I,--illumina Reads are from Illumina. Alters error rate used and adjusts for shorter reads
Filtering:
--clean Add a step to clean and detangle the pangraph
--max-covg INT Maximum coverage of reads to accept [default: 300]
Consensus/Variant Calling:
--genotype Add extra step to carefully genotype sites.
--kmer-avg INT Maximum number of kmers to average over when selecting the maximum likelihood path [default: 100]
Genotyping:
--local Needs: --genotype (Intended for developers) Use coverage-oriented (local) genotyping instead of the default ML path-oriented (global) approach.
-a INT Hard threshold for the minimum allele coverage allowed when genotyping [default: 0]
-s INT The minimum required total coverage for a site when genotyping [default: 0]
-D INT Minimum difference in coverage on a site required between the first and second maximum likelihood path [default: 0]
-F INT Minimum allele coverage, as a fraction of the expected coverage, allowed when genotyping [default: 0]
-E,--gt-error-rate FLOAT When genotyping, assume that coverage on alternative alleles arises as a result of an error process with rate -E. [default: 0.01]
-G,--gt-conf INT Minimum genotype confidence (GT_CONF) required to make a call [default: 1]
This will look for regions in the pangraph where the reads do not map
and attempt to locally assemble these regions to find novel variants.
The input file is the same as for compare - i.e. can handle multiple samples at a time.
$ pandora discover --help
Quasi-map reads to an indexed PRG, infer the sequence of present loci in the sample and discover novel variants.
Usage: src/ext/pandora discover [OPTIONS] <TARGET> <QUERY_IDX>
Positionals:
<TARGET> FILE [required] An indexed PRG file (in fasta format)
<QUERY_IDX> FILE [required] A tab-delimited file where each line is a sample identifier followed by the path to the fast{a,q} of reads for that sample
Options:
-h,--help Print this help message and exit
--discover-k INT:[0-32) K-mer size to use when discovering novel variants [default: 15]
--max-ins INT Max. insertion size for novel variants. Warning: setting too long may impair performance [default: 15]
--covg-threshold INT Positions with coverage less than this will be tagged for variant discovery [default: 3]
-l INT Min. length of consecutive positions below coverage threshold to trigger variant discovery [default: 1]
-L INT Max. length of consecutive positions below coverage threshold to trigger variant discovery [default: 30]
-d,--merge INT Merge candidate variant intervals within distance [default: 15]
-N INT Maximum number of candidate variants allowed for a candidate region [default: 25]
--min-dbg-dp INT Minimum node/kmer depth in the de Bruijn graph used for discovering variants [default: 2]
-v Verbosity of logging. Repeat for increased verbosity
Indexing:
-w INT Window size for (w,k)-minimizers (must be <=k) [default: 14]
-k INT K-mer size for (w,k)-minimizers [default: 15]
Input/Output:
-o,--outdir DIR Directory to write output files to [default: "pandora_discover"]
-t,--threads INT Maximum number of threads to use [default: 1]
--kg Save kmer graphs with forward and reverse coverage annotations for found loci
-M,--mapped-reads Save a fasta file for each loci containing read parts which overlapped it
Parameter Estimation:
-e,--error-rate FLOAT Estimated error rate for reads [default: 0.11]
-g,--genome-size STR/INT Estimated length of the genome - used for coverage estimation. Can pass string such as 4.4m, 100k etc. [default: 5000000]
--bin Use binomial model for kmer coverages [default: negative binomial]
Mapping:
-m,--max-diff INT Maximum distance (bp) between consecutive hits within a cluster [default: 250]
-c,--min-cluster-size INT Minimum size of a cluster of hits between a read and a loci to consider the loci present [default: 10]
Preset:
-I,--illumina Reads are from Illumina. Alters error rate used and adjusts for shorter reads
Filtering:
--clean Add a step to clean and detangle the pangraph
--clean-dbg Clean the local assembly de Bruijn graph
--max-covg INT Maximum coverage of reads to accept [default: 600]
Consensus/Variant Calling:
--kmer-avg INT Maximum number of kmers to average over when selecting the maximum likelihood path [default: 100]