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GRIDSS Post Somatic Software (GRIPSS)

GRIPSS applies a set of filtering and post processing steps on GRIDSS paired tumor-normal output to produce a high confidence set of somatic SV for a tumor sample. GRIPSS processes the GRIDSS output and produces a somatic vcf.

Repeat masker annotations must be included in the GRIDSS output before running GRIPSS. Details on how to include these are available on the GRIDSS readme here.

Usage

java -jar gripss.jar \
   -sample SAMPLE_T \
   -reference SAMPLE_N \
   -ref_genome /path/to/Homo_sapiens_assembly.fasta \
   -pon_sgl_file /path/to/gridss_pon_single_breakend.bed \
   -pon_sv_file /path/to/gridss_pon_breakpoint.bedpe \
   -known_hotspot_file /path/to/KnownFusionPairs.bedpe \
   -vcf /path/to/SAMPLE_T.gridss.unfiltered.vcf.gz \
   -output_dir /output_dir/

This will write 2 files:

  • SAMPLE_T.gridss.somatic.vcf.gz - all non-hard-filtered SVs
  • SAMPLE_T.gridss.somatic.filtered.vcf.gz - filtered for PASS and PON only

These two files are used in purple as the structural variant recovery vcf and structural variant vcf respectively.

The bed and bedpe files are available to download from HMFTools-Resources > GRIDSS for both GRCH 37 and 38.

Tumor-only mode

The reference argument is optional and if not supplied, GRIPSS will run in tumor-only mode in which case all filters that require the normal sample are de-activated. This includes minNormalCoverage, minRelativeCoverage, maxNormalSupport, shortSRNormalSupport, discordantPairSupport

Algorithm

There are 5 steps in GRIPSS described in detail below:

  1. Hard filters
  2. Realignment
  3. Soft filters
  4. Linkage, deduplication and rescue

1. Hard filters

Three hard filters are applied upfront before other processing occurs:

  • NO_MATE - Any non single breakend with no mate is filtered
  • MINIMUM_TUMOR_QUAL - Any variant with QUAL < 100 is filtered
  • MAX_NORMAL_SUPPORT - Any variant with normalSupport > min(max(3, 3% * tumorSupport), 8% * tumorSupport) is filtered as likely germline or artefact unless it links a pair of genes in the known pathogenic fusion list via translocation or local break junction of length more than 10kb. Ideally we would not allow any support for the variant in the normal, but contamination of the blood with tumor DNA is not uncommon.

2. Realignment

We realign imprecise variants where the breakend can be precisely resolved but the length of the insert sequence is unknown. By default GRIDSS offsets these variants by the uncertainty of the insert sequence with a wide CIPOS. GRIPSS realigns the variant to the earliest possible base in the uncertainty window which is the most likely base for the soft clipping.

For the purposes of backwards compatibility we also perform 3 other fixes to correct errors in earlier GRIDSS versions

  • Breakends are shifted to the centre of homology.
  • Ensure that breakends pairs are internally consistent in their positions
  • Ensure that local and remote inexact homology are internally consistent.

3. Soft filters

The following filters are applied to all variants

Filter Default Description / purpose
minQual 400 (single breakend:500) Minimum absolute tumor support for variant
minNormalCoverage 8 Variants with low coverage in germline may be germline variants.
maxNormalRelativeSupport 0.03 Reads supporting variant in the normal sample may not exceed 3% of read support in the tumor.
minTumorAF 0.005 Low AF variants in high depth regions may be artefacts
imprecise FALSE Imprecise variants may be artefacts linking low mappability regions of the genome.
discordantPairSupport TRUE Variants (except for DEL,INS & DUP < 1000 bases) must have at least 1 read mapped at each end. Avoids artefacts linking regions of low mappability. Not suitable for non paired reads or very short fragment sizes. Single breakends without any assembly read pairs (BASRP=0) are also filtered
PON FALSE Breakpoint must be found < 3 times in our cohort in ~3800 germline samples (panel of normals). The PON excludes imprecise calls and breakpoints <75 qual score and breakends < 428 qual score. MH is counted in overlap and a 2bp margin of error is allowed for.
maxPolyAHomLength 6 Variants with long POLYA homology are frequent artefacts at low VAF
maxPolyGLength 16 Long stretches of polyG/polyC are extremely rare in the ref genome but are known sequencer artefacts. Single breakends with insert sequences containing long polyG homopolymers are filtered. This filter is also applied to break junctions where 1 end maps in the POLY-G region of LINC00486 (v38: chr2:32,916,190-32,916,630; v37: 2:33,141,260-33,141,700).

We also have 6 special filters applying to specific short variant categories:

Filter Default Scope Description
minLength 32 DEL, DUP & INS Minimum absolute length (including insert sequence length) for short DEL and DUP SV to be called.
maxHomLengthShortInv 6 INV(<40b) Very short INV with high homology are a common sequencer artefact
shortStrandBias TRUE INS,DEL & DUP(<1kb) Short DEL and DUP must be strand balanced
shortSRTumorSupport TRUE INS,DEL & DUP(<1kb) Short DELs and DUPs must be supported by at least 1 split read or in the case of very short DEL and INS at least 1 supporting indel containing read.
shortSRNormalSupport FALSE INS,DEL & DUP(<1kb) Short DELs and DUPs must not be supported by 1 split read or 1 indel containing read in the normal
shortDelInsArtefact DEL(<1kb) Filter any short DEL where the insert sequence length + 1 = deletion length, unless the insert sequence is identical to the reverse complement (ie a short reciprocal inversion). This is a known GRIDSS artefact.

4. Linkage, deduplication and rescue

A. Assembly linkage

Variants located on a single assembly are given a unique 'asm' identifier.

B. Deduplication and transitive linkage

The GRIDSS output may contain structural variants which may be duplicated either by a single SV or by a chain of SVs with breakends proximate to each other which may or may not already be linked by assembly. In the case where a variant is duplicated by a chain, we term this variant the spanning variant and these links to be transitive links.

For a variant to be marked as a duplicate, we must find 2 candidate transitive breakends which match the orientation and position of the spanning variant, within CIPOS bounds and allowing for the insert sequence length. The CIPOS bounds for imprecise variants are ignored for this purpose and the match must be exact (see realignment section above).

The candidate transitive breakends must be linkable in a continuous chain as one of the following cases:

  • same variant - opposite breakends of the same SV
  • same assembly - the 2 transitive SVs are part of the same assembly and oriented away from each other
  • 1 transitive jump - the far breakend of the 2 transitive SVs / assemblies face each other and each link must be less than 1000 bases
  • 2 transitive jumps - the far breakend of the 2 transitive SVs / assemblies both face opposite ends of a 3rd SV or assembly and each link must be less than 1000 bases

As a spanning variant may have multiple alternate paths, we first consider only assembly linked paths favouring those with the fewest jumps.
If an assembly-only solution is unavailable we include up to two transitive jumps, but this must result in a single alternate path. If there are multiple alternate paths with transitive links, none will be selected.

If the deduplicated spanning variant is PRECISE, then the length of the insert sequence of the spanning variant must match the entire chain length of the transitive variants (again allowing for CIPOS bounds and insert sequence length of precise variants).

Any single breakend which matches the position and orientation of another breakend or breakjunction (within CIPOS bounds) is also filtered as DEDUP. GRIPSS prioritises retaining breakends / breakjunctions linked by assembly or transitive link, then breakends that are passing and finally by highest qual score.

C. Linkage by double stranded break

Double stranded break sites can lead to 2 proximate breakends in very close proximity with opposite orientation and either a small gap in between or a small overlap, frequently <30 bases. GRIPSS links breakends with a unique 'DSB' id where there is one and only one (non-DEDUPed) breakend within 30 bases (allowing for CIPOS bounds) with opposite orientation and both breakends have qual > 100.

D. Rescue

Any breakend that is linked to a PASS breakend (by one of the 3 above rules) and is NOT filtered as DEDUP is rescued from soft filtering and marked as PASS. Breakend pairs that link a pair of genes to make a known pathogenic fusions are also rescued for all soft filters except maxPolyAHomLength.

To improve detection of mobile element insertions, we also rescue pairs of breakends or breakjunctions which are linked by ‘DSB’ and NOT PON filtered, with combined qual > 1000 and with at least one of the breakends having the characteristic poly-A insert sequence tail of a mobile element insertion. We define a poly-A tail as either at least 16 of the last 20 bases of the sequence are A or there is a repeat of 10 or more consecutive A or within the last 20 bases of the insert sequence. At the insertion site, negative oriented breakends must have poly-A tails at the end of the insert sequence and positive oriented breakends must have poly-T at the start of the insert sequence (if inserted on the reverse strand).

Note that for DSB and hotspot rescue, neither the rescued variant nor the rescuing variant is permitted to be a DEL, INS or DUP < 10kb in length.

Version History and Download Links

  • 1.11
    • Fix hotspot matching
    • Replace other ambiguous bases in bam with N
    • Fixed description of TAF: Tumor allelic frequency (fragment support / total support)
    • Do not attempt transitive linking if there is 500,000+ variants
    • Changed default value of hardMaxNormalRelativeSupport from 0.06 to 0.08
  • 1.10
    • Removed viral insertion exception from normal relative support filter
    • Add EventType flag [DEL, INS, DUP, INV, SGL, BND]
    • Check IC flag whenever we check SR
  • 1.9
    • Warn if malformed BEALN field in input
    • Only include specified samples in VCF output
    • tumor argument is now mandatory
    • reference argument is still optional but excluding it will run in tumor-only mode
    • HardMaxNormalAbsoluteSupport does not trigger unless SoftMaxNormalRelativeSupport does also
  • 1.8
    • Legs of variant always have same filters
  • 1.7
    • Fix bug in BEALN interpretation
  • 1.6
    • Fix bug where alt does not always realign
  • 1.5
    • Tumor only support
    • Added GripssHardFilterApplicationKt application
  • 1.4
    • Fix bug when trying to use non-default parameter values
  • 1.3
    • Fixed bug to allow output VCF name to be relative path
    • Fixed bug handling contigs with colons eg HLA-A*23:01:01
  • 1.2
    • Hotspots cannot recover HardMinQualFiltered variants
    • Ignore maxNormalRelativeSupport filter for single breakends with viral sequence alignments
    • BEALN, INSRMRC and INSRMRT are now mandatory requirements
  • 1.1
    • Added HardMinTumorQual [100] filter
    • Added HardMaxNormalAbsoluteSupport [3] filter
    • Added HardMaxNormalRelativeSupport [0.06] filter to replace MaxNormalRelativeSupport [0.03]
    • Added SoftMaxNormalRelativeSupport [0.03] filter
    • Qual filters and QUAL field in output VCF now refer to tumor qual only
    • Added optional reference and tumor parameters
    • Rescue DSB linked mobile element insertions
  • 1.0
    • Initial Release