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PfINV

Detection of inversions in P. falciparum from long read assemblies.

Overview

This repo accompanies an upcoming publication describing the identification of inversions in long read P. falciparum assemblies. It contains the core inversion detection script (src/), its support scripts (support/), and a number of scripts associated with the manuscript itself (manuscript/). The core discovery program is a command line Unix tool, built in bash and tested in both Linux and MacOS environments. It relies on nucmer, which can be automatically installed with setup.sh.

Quick Start

# first time setup
bash setup.sh

# discovery with a list of sample assemblies (one path per line in samples.txt)
bash findINVs.sh -r=ref.fasta -s=samples.txt

Method

Inversion detection relies upon nucmer alignment of a candidate fasta (the 'sample') against a reference fasta. Mis-alignments are then examined with show-coords, with those regions that align in reverse being identified as inversions. Aligned regions below a minimum size (default 300 bp) or whose relative bp coordinates (between the reference and sample assemblies) exceed a maximum distance (default 50,000 bp) will be discarded.

Core Algorithm

  1. show-coords -HT ${SAMPLE}.delta: Call show-coords on post-nucmer sample-on-reference alignment.
  2. awk -F'\t' '$3 > $4': Filter to aligned regions whose sample start location exceeds its sample end location (ie. it is in reverse alignment).
  3. sed 's,'"$REFname"'_,'"$SAMPLE"'_,g': Rename reference chromosome to sample's name for later matching.
  4. awk -F'\t' '$8 == $9': Exclude alignments in different chromosomes.
  5. sort -r -k5n: Reverse sort alignments by the sample sequence length.
  6. awk -v minSize="$MIN_SIZE" -F'\t' '$5 >= minSize': Remove alignments below or equal to a minimum size.
  7. awk -v maxDist="$MAX_DISTANCE" -F'\t' 'function abs(v) {return v < 0 ? -v : v} {if (abs($2 - $3) <= maxDist) {print $0}}': Remove alignments above a maximum relative distance (coords in sample vs reference).
  8. cut -f1-8: Remove the final (now duplicate) column.
  9. > ${SAMPLE}.INV: Write inversions to sample.INV.

Output

Following a successful run findINVs.sh will output one .INV file per sample, in which each row represents an inversion. This file is formatted as:

StartRef EndRef StartSample EndSample LengthRef LengthSample Identity SampleChrom
Reference start position (bp) Reference end position (bp) Sample start position (bp) Sample end position (bp) Length in reference Length in sample Percentage Identity Chromosome

Specific Scripts

src/

  • setup.sh Run to ensure that all required packages are installed. This will prompt to run as sudo.
  • findINVs.sh Core detection pipeline, run as findINVs.sh -h to return the help page. Note that input fastas should be named <sample>.fasta, and chromosome or contig names should be formatted as <sample>_<chromosome>. Input arguments include:
    • -r --ref (reference file) The path to your reference genome (.fasta).
    • -s --samples (sample list) The path to a textfile containing paths (one per line) to your sample fasta files.
    • -m --minsize (min. size) The minimum size for inversions, any below this cut-off will be discarded.
    • -d --maxdist (max. distance) The maximum relative distance in bp coordinates between your reference and candidate fastas.

support/

  • grabRef.sh Download the references used in the manuscript.
  • embl2fasta.py Convert embl files to fasta format (for input to findINVs.sh).

manuscript/

Scripts used to create the figures in the manuscript, free for use and adaptation by the wider community. Their filenames are indicative of their function.

Raising Issues

If you find bugs, please raise them as an issue on github. For questions regarding the manuscript, email me at [email protected].