Output

Analyse genome sizes

This command generates data/04_rename_genome/genome_size_stats.csv as output. This csv file has the following fields:

  • assembly: name of the assembly, as specified in data/data.csv
  • ancestor: name of the ancestor, as specified in data/data.csv
  • size_assembly: size of the assembly, in bp
  • size_ancestor: size of the ancestor, in bp
  • difference: size_assembly - size_ancestor. This value is negative when the ancestor is larger than the assembly
  • percent_change: the difference in size, as a percent of the size of the assembly

See a sample table in the tutorial.

Predict SVs

mummer4 is used to perform alignments between assembly-ancestor pairs. The alignment files are stored in data/06_nucmer_alignment/<assembly>/ directories. Each of these directories contains the following files:

  • <assembly>.delta: The all-vs-all alignment generated by the nucmer command of mummer4
  • <assembly>.delta.filter: The <assembly>.delta file is filtered by the delta-filter command to only retain alignments > 95% sequence identity and > 100bp long.
  • <assembly>.filtered.coords: A file summarising information about each delta alignment in the filtered file.

For specific information about these file formats, please refer to the mummer manual.

Next SyRI is used to call SVs using those alignments. The output files for each assembly is in the respective data/07_syri_output/<assembly>/ directories. Each of these directories contains the following files:

  • <assembly>syri.out: a tsv file that describes the location and type of SVs in the assembly, relative to its ancestor. We refer the user to the official SyRI documentation for information on interpreting this table. You may notice that the reference and query chromosome ID read as "genome". This is because SyRI attempts to call variants between homologous chromosomes, and hence expects the reference and query ID to be the same string. To prevent this error from occurring, the genomes are renamed to a placeholder string 'genome'. You may also see that for a deletion, the query start and stop site are the same. You can also find a sample table in the tutorial.
  • <assembly>syri.vcf: a variant calling format (vcf) file describing the same SVs

Raw output generated by SyRI was refined with custom python scripts to predict a minimal set of structural variants. Briefly, new insertions of individual IS elements due to transposition events were filtered out, ambiguous nomenclature for deletions was replaced with consistent terminology, falsely classified duplications were corrected, adjacent deletions were merged, and adjacent syntenic regions were merged. This filtered set of SVs is a tsv file data/07_syri_output/<assembly>/<assembly>_clean.syri.out and follows the same file format as <assembly>syri.out.

Finally, a modified version of plotsr is used to generate synteny plots. Two synteny plots are generated in data/07_syri_output/<assembly>/:

  • <assembly>.plot.pdf: synteny plot corresponding to the SVs in <assembly>syri.out
  • <assembly>.plot.2.pdf: synteny plot corresponding to SVs in <assembly>_clean.syri.out

Tip: The 4th and 5th columns of the <assembly>_clean.syri.out file have the reference/query sequences for indels. For large deletions, these field can have thousands of bases. To view this tsv file in the terminal without these columns, try running

cat <assembly>_clean.syri.out | cut --complement -f 4-5

Note: SyRI sometimes fails to correctly identify copy number variation (deletions and amplifications) or more complex rearrangements (such as overlapping inversions).

Annotate genes in SVs

This command uses prokka annotate genes in the assemblies and combines insertion sequence ore and generates GFF files for each in data/09_annotated_genomes/<assembly>/<assembly>.gff.

Next, breseq uses these GFF files to annotate the genes present in the deletions, inversions and amplifications. This step produces two outputs for each assembly:

  • data/12_genome_diff_tables/gd/<assembly>.gd: This is a GenomeDiff format that is a tsv file describing the locations, size and types of SVs. Please see the breseq documentation for more information about this file format.
  • data/12_genome_diff_tables/html/<assembly>.html: This is an html, human-readable version of the GenomeDiff file that contains the names of the genes contained in the SV regions. For an example of this table, please see the tutorial. For information about the columns in this table, please see the breseq documentation.

Predict SV mechanism

This command annotates the insertion sequences at the boundaries of structural variants (for deletions and inversions only) and predicts putative mechanisms. All of the output from this step is in the directory data/11_annotated_boundaries/. Each assembly has three files associated with it:

  • data/11_annotated_boundaries/<assembly>_boundaries.csv: This csv file annotates insertion sequences that are within a threshold distance of the start and end sites of a structural variant. The threshold has arbitrarily been set to 20bp. This csv file has the following columns. Note: For deletions, the query_start and query_stop coordinates will be of the same length. Additionally, the distances can be positive or negative, depending on if the insertion sequence starts/ends before or after that edge of the SV.

    • ref_start: Start site of the SV in the ancestor
    • ref_stop: Stop site of the SV in the ancestor
    • query_start: Start site of the SV in the assembly
    • query_stop: Stop site of the SV in the aseembly
    • tag_3: Type of SV. This follows the SyRI nomenclature (SYN: syntenic region, INV: inversion, DUP: duplication, TRANS: translocation, DEL: deletion)
    • L_ref: Name of insertion sequence within threshold distance of the start site of the SV in the ancestor. This is 'NA' if none is found.
    • L_ref_distance: Distance of the closest end of the insertion sequence from the start site of the SV in the ancestor.
    • R_ref: Name of insertion sequence within threshold distance of the stop site of the SV in the ancestor. This is 'NA' if none is found.
    • R_ref_distance: Distance of the closest end of the insertion sequence from the start site of the SV in the ancestor.
    • L_query: Name of insertion sequence within threshold distance of the start site of the SV in the assembly. This is 'NA' if none is found.
    • L_query_distance: Distance of the closest end of the insertion sequence from the start site of the SV in the assembly.
    • R_query: Name of insertion sequence within threshold distance of the stop site of the SV in the assembly. This is 'NA' if none is found.
    • R_query_distance: Distance of the closest end of the insertion sequence from the start site of the SV in the assembly.

  • data/11_annotated_boundaries/<assembly>_deletion.csv: This file contains the same fields as above, with two additional columns. These columns describe the various configurations in which insertion sequences can flank a deletion.

    • Mechanism: This field can take on values of 'between_IS' (when a deletion occurs between two IS elements that were already present in the ancestor), 'IS_mediated' (when only one or none of the implicated IS elements were present in the ancestor) and 'other' (through recombination between other repeats or illegitimate recombination)
    • Evidence: This field can take on values of 'full', 'evolved', 'incomplete' or 'NA'.

The following image describes the different configurations in which insertion sequences can be present around a deletion, and the mechanism and evidence that is assigned on that basis. The red arrow depicts the region that was deleted, and the blue boxes indicate insertion sequences.

image_deletion

  • data/11_annotated_boundaries/<assembly>_inversion.csv: This file describes the various configurations in which insertion sequences can flank an inversion. It contains the same columns as the above file.

The following image describes the different configurations in which insertion sequences can be present around an inversion, and the mechanism and evidence that is assigned on that basis. The orange arrow depicts the region that was inverted, and the blue boxes indicate insertion sequences.

image_inversion

Additionally, this command generates two summary files: data/11_annotated_boundaries/deletion_mechanism.csv and data/11_annotated_boundaries/inversion_mechanism.csv. They contain information about the mechanism of deletions and inversions respectively, for all of the assemblies. Both these files have the same fields:

  • clone: name of the assembly
  • total: total number of inversions or deletions
  • between_IS: number of inversions or deletions that whose mechanism was 'between_IS'
  • IS_mediated: number of inversions or deletions that whose mechanism was 'IS_mediated'
  • other: number of inversions or deletions whose mechanism was through recombination between other repeats or illegitimate recombination

For examples of all of these tables, please see the tutorial.

Predict replichore and inversion balance

This command analyses the length of the two replichores of each assembly, and classifies inversion as inter-replichore or intra-replichore. It produces the following output:

  • data/08_reindex_genome_oric/replichore_arms.csv: a csv file that describes the length of the two replichores for all of the assemblies. It contains the following fields:

    • clone: Name of the assembly
    • ori: Location of the oriC sequence. You may notice that this is 0 for all of the assemblies. This is because the circular genomes have been rotated for this step to start at the oriC to normalise it.
    • dif: Location of the dif sequence. This coordinate represents the first base at which the supplied dif site starts.
    • length: Total length of the genome of the ancestor
    • arm_1: Length of one replichore (in this case, the distance in bp from the oriC to the dif site)
    • arm_2: Length of the other replichore (in this case, the distance in bp from the dif site to end of the genome)
    • ratio: The ratio of the longer arm to the shorter arm. This value should be >=1
    • percent: The percent of the total length of the genome contained in the longer arm. For a perfectly balanced genome, this value is 50 and gets larger as the genome gets more imbalanced. It is never less than 50.

  • data/11_annotated_boundaries/inversion_replichores.csv: a csv file that tallies the count of each type of inversion (inter-replichore vs intra-replichore) for all of the assemblies.

    • clone: Name of the assembly
    • total_inversions: Total number of inversions detected in that assembly. This is the sum of the total_inter_replichore and total_intra_replichore.
    • across_ori: Number of inversions that were across the oriC site
    • across_dif: Number of inversions that were across the dif site
    • total_inter_replichore: Total number of inter-replichore inversions. This is the sum of across_ori and across_dif
    • total_intra_replichore: Total number of inter-replichore inversions.

  • data/11_annotated_boundaries/<assembly>_inversion_classification.csv: a csv file that has more detailed information about all of the inversions in a given assembly. It has the same fields as data/11_annotated_boundaries/<assembly>_inversion.csv (see Predict SV mechanism), with three additional fields:

    • classification: Describes the type of inversion, and takes on values 'across_ori', 'across_dif', 'intra_replichore'.
    • length: Length of the inversion in bp.
    • symmetry_percent: The percent of the total length of the inversion contained in the longer arm. For a perfectly symmetric inversion, this value is 50 and gets larger as the inversion gets more asymmetric. It is never less than 50.

  • data/11_annotated_boundaries/inversion_replichores_long.csv: a csv file that contains a list of all inversions across all assemblies. It contains the following fields:

    • clone: Name of the assembly
    • classification: Describes the type of inversion, and takes on values 'across_ori', 'across_dif', 'intra_replichore'.
    • length: Length of the inversion in bp.
    • mechanism: Putative mechanism of the inversion, and takes on values 'between_IS', 'IS_mediated' and 'other' See Predict SV mechanism above.
    • symmetry_percent: The percent of the total length of the inversion contained in the longer arm. For a perfectly symmetric inversion, this value is 50 and gets larger as the inversion gets more asymmetric. It is never less than 50.