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Pandora is a tool for bacterial genome analysis without using a reference genome, including genetic variation from SNPs to gene presence/absence across the whole pan-genome. Core ideas are:

• new samples look like recombinants (plus mutations) of things seen before
• we should be analysing nucleotide-level variation everywhere, not just in core genes
• arbitrary reference genomes are unnatural

Pandora works with Illumina or nanopore data, allowing per-sample analysis (sequence inference and SNP/indel/gene-calling) and comparison of multiple samples. To do this it uses population reference graphs (PRG) which have been built for orthologous blocks of interest (e.g. genes and intergenic regions). See https://github.com/rmcolq/make_prg for a pipeline which can construct these PRGs from a set of aligned sequence files.

It can do the following for a single sample (read dataset):

• Output inferred gene sequences for the orthologous chunks (eg genes) in the PRG
• Output a VCF showing the variation found in the pangenome genes which are present, with respect to any reference in the PRG.

For a collection of samples, it can:

• Output a matrix showing inferred copy-number of each gene in each sample genome.

• Output one VCF per orthologous-chunk, showing how samples which contained this chunk differed in their gene sequence. Variation is shown with respect to the most informative recombinant path in the PRG . Soon, in a galaxy not so far away, it will allow

• discovery of new variation not in the PRG

Warning - this code is still in development.

• Requires a Unix or Mac OS.

• Requires a system install of zlib. If this is not already installed, this tutorial is helpful.

• Requires a system installation of boost containing the system, filesystem, log (which also depends on thread and date_time) and iostreams libraries. If not already installed use the following or look at this guide.

  wget https://sourceforge.net/projects/boost/files/boost/1.62.0/boost_1_62_0.tar.gz --no-check-certificate
  tar xzf boost_1_62_0.tar.gz
  cd boost_1_62_0
  ./bootstrap.sh [--prefix=/prefix/path] --with-libraries=system,filesystem,iostreams,log,thread,date_time
  ./b2 install
  

• Download and install pandora as follows:

  git clone https://github.com/rmcolq/pandora.git
  cd pandora
  mkdir build
  cd build
  cmake [-DCMAKE_PREFIX_PATH=/prefix/path] ..
  make
  ctest -VV
  cd ..
  

Instead you can download and use the singularity container:

singularity pull --force --name pandora.simg shub://rmcolq/pandora:pandora
singularity exec pandora.simg pandora

Pandora assumes you have already constructed a fasta-like file of graphs, one entry for each gene/ genome region of interest.

Takes a fasta-like file of PRG sequences and constructs an index, and directory of gfa files to be used by pandora map.

  Usage: pandora index [options] <prgs.fa>
  Options:
  	-h,--help			Show this help message
  	-w W				Window size for (w,k)-minimizers, default 14
  	-k K				K-mer size for (w,k)-minimizers, default 15

The index stores (w,k)-minimizers for each PRG path found. These parameters can be specified, but default to w=1, k=15.

This takes a fasta of noisy long read sequence data and compares to the index. It infers which of the PRG genes/elements is present, and for those that are present it outputs the inferred sequence.

  Usage: pandora map -p PRG_FILE -r READ_FILE -o OUTDIR <option(s)>
  Options:
   -h,--help			 Show this help message
   -p,--prg_file PRG_FILE	 Specify a fasta-style prg file
   -r,--read_file READ_FILE	 Specify a file of reads in fasta format
   -o,--outdir OUTDIR	         Specify directory of output
   -w W				 Window size for (w,k)-minimizers, must be <=k, default 14
   -k K				 K-mer size for (w,k)-minimizers, default 15
   -m,--max_diff INT		 Maximum distance between consecutive hits within a cluster, default 500 (bps)
   -e,--error_rate FLOAT	 Estimated error rate for reads, default 0.11
   --genome_size NUM_BP	         Estimated length of genome, used for coverage estimation
   --output_kg			 Save kmer graphs with fwd and rev coverage annotations for found localPRGs
   --output_vcf			 Save a vcf file for each found localPRG
   --vcf_refs REF_FASTA		 A fasta file with an entry for each LocalPRG giving reference sequence for
                                 VCF. Must have a perfect match in the graph and the same name as the graph
   --illumina			 Data is from illumina rather than nanopore, so is shorter with low error rate
   --bin			 Use binomial model for kmer coverages, default is negative binomial
   --max_covg			 Maximum average coverage from reads to accept
   --regenotype			 Add extra step to carefully genotype SNP sites