GenomeDiff Format

The GenomeDiff file format describes mutational differences between a reference DNA sequence and a sample. It may also include evidence from computational analysis or experiments that supports mutations.

An example of a portion of a GenomeDiff file:

#=GENOME_DIFF 1.0
DEL  61      11      NC_001416       139     1
INS  62      12      NC_001416       14266   G
SNP  63      13      NC_001416       20661   G
INS  64      14      NC_001416       20835   C
SNP  65      15      NC_001416       21714   A
DEL  60      33,1    NC_001416       21738   5996
SNP  66      35      NC_001416       31016   C
...
MC   9               NC_001416       1       2       0       0       left_inside_cov=0       left_outside_cov=NA     right_inside_cov=0      right_outside_cov=169
RA   11              NC_001416       139     0       G       .       frequency=1     new_cov=34/40   quality=309.0   ref_cov=0/0     tot_cov=34/40
JC   2               NC_001416       5491    1       NC_001416       30255   1       0       alignment_overlap=4     coverage_minus=8        coverage_plus=0 flanking_left=35        flanking_right=35       key=NC_001416__5491__1__NC_001416__30251__1__4____35__35__0__0  max_left=30     max_left_minus=30       max_left_plus=0 max_min_left=0  max_min_left_minus=0    max_min_left_plus=0     max_min_right=11        max_min_right_minus=11  max_min_right_plus=0    max_right=11    max_right_minus=11      max_right_plus=0        min_overlap_score=44    pos_hash_score=7        reject=NJ,COV   side_1_annotate_key=gene        side_1_overlap=4        side_1_redundant=0      side_2_annotate_key=gene        side_2_overlap=0        side_2_redundant=0      total_non_overlap_reads=8       total_reads=8
JC   3               NC_001416       13180   1       NC_001416       13218   1       0       alignment_overlap=4     coverage_minus=1        coverage_plus=0 flanking_left=35        flanking_right=35       key=NC_001416__13180__1__NC_001416__13214__1__4____35__35__0__0 max_left=17     max_left_minus=17       max_left_plus=0 max_min_left=0  max_min_left_minus=0    max_min_left_plus=0     max_min_right=14        max_min_right_minus=14  max_min_right_plus=0    max_right=14    max_right_minus=14      max_right_plus=0        min_overlap_score=14    pos_hash_score=1        reject=NJ,COV   side_1_annotate_key=gene        side_1_overlap=4        side_1_redundant=0      side_2_annotate_key=gene        side_2_overlap=0        side_2_redundant=0      total_non_overlap_reads=1       total_reads=1
RA   12              NC_001416       14266   1       .       G       frequency=1     new_cov=44/31   quality=186.3   ref_cov=0/0     tot_cov=44/31
JC   5               NC_001416       14869   -1      NC_001416       15609   -1      0       alignment_overlap=7     coverage_minus=1        coverage_plus=0 flanking_left=35        flanking_right=35       key=NC_001416__14869__0__NC_001416__15616__0__7____35__35__0__0 max_left=21     max_left_minus=21       max_left_plus=0 max_min_left=0  max_min_left_minus=0    max_min_left_plus=0     max_min_right=7 max_min_right_minus=7   max_min_right_plus=0    max_right=7     max_right_minus=7       max_right_plus=0        min_overlap_score=7     pos_hash_score=1        reject=NJ,COV   side_1_annotate_key=gene        side_1_overlap=7        side_1_redundant=0      side_2_annotate_key=gene        side_2_overlap=0        side_2_redundant=0      total_non_overlap_reads=1       total_reads=1

Format specification

Version line

The first line of the file must define that this is a GenomeDiff file and the version of the file specification used:

#=GENOME_DIFF 1.0

Metadata lines

Lines beginning with #=<name> <value> are interpreted as metadata. (Thus, the first line is assigning a metadata item named GENOME_DIFF a value of 1.0.) Names cannot include whitespace characters. Values may include whitespace characters. Lines with the same name are concatenated with single spaces added between them.

Comment lines

Lines beginning with whitespace and # are comments. Comments may not occur at the end of a data line.

Data lines

Data lines describe either a mutation or evidence from an analysis that can potentially support a mutational event. Data fields are tab-delimited. Each line begins with several fields containing information common to all types, continues with a fixed number of type-specific fields, and ends with an arbitrary number of name=value pairs that store optional information.

1. type <string>

   type of the entry on this line.

2. id or evidence-id <uint32>

   For evidence and validation lines, the id of this item. For mutation lines, the ids of all evidence or validation items that support this mutation. May be set to ‘.’ if a line was manually edited.

3. parent-ids <uint32>

   ids of evidence that support this mutation. May be set to ‘.’ or left blank.

mutation types are 3 letters: SNP, SUB, DEL, INS, MOB, AMP, CON, INV.

evidence types are 2 letters: RA, MC, JC, UN.

validation types are 4 letters: TSEQ, PFLP, RFLP, PFGE, PHYL, CURA.

Mutational Event Types

SNP: Base substitution mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. new_seq <char>

   new base at position

SUB: Multiple base substitution mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position of the first replaced nucleotide in reference sequence fragment.

6. size <uint32>

   number of bases after the specified reference position to replace with new_seq

7. new_seq <string>

   new bases at position

DEL: Deletion mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. size <uint32>

   number of bases deleted in reference, including reference position.

INS: Insertion mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment, after which the INS is placed.

6. new_seq <string>

   new base inserted after the specified reference position

MOB: Mobile element insertion mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. repeat_name <string>

   name of the mobile element. Should correspond to an annotated repeat_region in the reference.

7. strand <1/-1>

   strand of mobile element insertion.

8. duplication_size <uint32>

   number of bases duplicated during insertion, beginning with the specified reference position.

AMP: Amplification mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. size <uint32>

   number of bases duplicated starting with the specified reference position.

7. new_copy_number <uint32>

   new number of copies of specified bases.

CON: Gene conversion mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment that was the target of gene conversion from another genomic location.

6. size <uint32>

   number of bases to replace in the reference genome beginning at the specified position.

7. region <sequence:start-end>

   Region in the reference genome to use as a replacement.

INV: Inversion mutation

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. size <uint32>

   number of bases in inverted region beginning at the specified reference position.

Standard name=value pairs

Counting Mutations

These attributes control how molecular events in a a GenomeDiff are counted for summary purposes.

• between=<element_name>

  This mutation occurs between copies of this element. For example, a deletion caused by recombination between two copies of a mobile element.

• mediated=<element_name>

  This mutation was mediated by insertion of a new copy of this element and recombination with an existing copy, such that the number of this element did not net increase in the resulting genome.

• adjacent=<element_name>

  This mutation

• with=<mutatiion_id>

  This mutation should be counted as a single molecular event with the other specified mutation. For example, active mobile elements may lose and gain a few bases at their margins, and when this occurs the most parsimonious explanation is one round of recombination or excision and re-insertion.

Applying Mutations

These attributes control how mutations are applied when building a new reference genome from the original reference genome and a GenomeDiff and when building phylogenetic trees between multiple samples. They are not generated automatically by breseq.

• before=<mutation_id> or after=<mutation_id>

  Apply this mutation before or after another mutation. For example, did a base substitution occur after a region was duplicated, thus it is only in one copy or did it occur before the duplication, thus altering both copies? Did a base substitution happen before a deletion, hiding a mutation that should be included in any phylogenetic inference? The before. When neither of these attributes is present, mutations will be applied in the order in which they appear in the file.

• within=<mutation_id>, within_position=<mutation_id>, within_copy=<mutation_id>

  This mutation happens inside of a different mutation. These options can specify, for example, that a base substitution happens in the second copy of a duplicated region. within and within_position must both be provided if one is supplied. If within_copy is not provided (because it is unknown), the mutation will be placed arbitrarily in the first copy. Note that the actual position of this mutation is still used for annotating its effects.

Evidence Types

RA: Read alignment evidence

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. insert_position <uint32>

   number of bases inserted after the reference position to get to this base. An value of zero refers to the base. A value of 5 means that this evidence if for the fifth newly inserted column after the reference position.

7. ref_base <char>

   base in the reference genome.

8. new_base <char>

   new base supported by read alignment evidence.

MC: Missing coverage evidence

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. start <uint32>

   start position in reference sequence fragment.

6. end <uint32>

   end position in reference sequence of region.

7. start_range <uint32>

   number of bases to offset after the start position to define the upper limit of the range where the start of a deletion could be.

8. end_range <uint32>

   number of bases to offset before the end position to define the lower limit of the range where the start of a deletion could be.

Essentially this is evidence of missing coverage between two positions in the ranges [start, start+start_range] [end-end_range, end].

JC: New junction evidence

4. side_1_seq_id <string>

   id of reference sequence fragment containing side 1 of the junction.

5. side_1_position <uint32>

   position of side 1 at the junction boundary.

6. side_1_strand <1/-1>

   direction that side 1 continues matching the reference sequence

7. side_2_seq_id <string>

   id of reference sequence fragment containing side 2 of the junction.

8. side_2_position <uint32>

   position of side 2 at the junction boundary.

9. side_2_strand <1/-1>

   direction that side 2 continues matching the reference sequence.

9. overlap <uint32>

   Number of bases that the two sides of the new junction have in common.

UN: Unknown base evidence

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. start <uint32>

   start position in reference sequence of region.

6. end <uint32>

   end position in reference sequence of region.

Validation Types

These items indicate that mutations have been validated by further, targeted experiments.

CURA: True-positive curated by an expert

An expert has examined the data output from a prediction program and determined that this mutations is a true positive.

Line specification:

4. expert <string>

   Name or initials of the person who predicted the mutation.

FPOS: False-positive curated by an expert

An expert has examined the raw read data and determined that this predicted mutation is a false positive.

Line specification:

4. expert <string>

   Name or initials of the person who predicted the mutation.

PHYL: Phylogenetic comparison

This validation was transferred from validation in another, related genome.

Line specification:

4. gd <string>

   Name of the genome_diff file containing the evidence.

TSEQ: Targeted re-sequencing

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. primer1_start <uint32>

   position in reference sequence of the 5’ end of primer 1.

6. primer1_end <uint32>

   position in reference sequence of the 3’ end of primer 1.

7. primer2_start <uint32>

   position in reference sequence of the 5’ end of primer 2.

8. primer2_end <uint32>

   position in reference sequence of the 3’ end of primer 2.

For primer 1, start < end. For primer 2, end < start.

PFLP: PCR-fragment length polymorphism

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. primer1_start <uint32>

   position in reference sequence of the 5’ end of primer 1.

6. primer1_end <uint32>

   position in reference sequence of the 3’ end of primer 1.

7. primer2_start <uint32>

   position in reference sequence of the 5’ end of primer 2.

8. primer2_end <uint32>

   position in reference sequence of the 3’ end of primer 2.

For primer 1, start < end. For primer 2, end < start.

RFLP: Restriction fragment length polymorphism

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. primer1_start <uint32>

   position in reference sequence of the 5’ end of primer 1.

6. primer1_end <uint32>

   position in reference sequence of the 3’ end of primer 1.

7. primer2_start <uint32>

   position in reference sequence of the 5’ end of primer 2.

8. primer2_end <uint32>

   position in reference sequence of the 3’ end of primer 2.

9. enzyme <string>

   Restriction enzyme used to distinguish reference from mutated allele.

For primer 1, start < end. For primer 2, end < start.

PFGE: Pulsed-field gel electrophoresis

Changes in fragment sizes of genomic DNA digested with restriction enzymes and separated by pulsed-field

Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. restriction enzyme <string>

Restriction enzyme used to digest genomic DNA and observe fragments.

NOTE: Note

Generic container for a note about a mutation prediction

Line specification:

4. note <string>

   Free text note.

MASK: Repeat mask a section

Artificially mask a section of DNA as “N”s. This is useful for creating fake reference sequences. Particularly for targeted sequencing approaches. Line specification:

4. seq_id <string>

   id of reference sequence fragment containing mutation, evidence, or validation.

5. position <uint32>

   position in reference sequence fragment.

6. size <uint32>

   number of bases masked to “N” in reference, including reference position.