See the Lilac Google doc
To install, download the latest compiled jar file from the download links.
Lilac requires the following resource files:
- Ref Genome FASTA
- HLA allele nucleotide and amino acid definitions, generated by Lilac (see below)
Reference files are available here HMFTools-Resources:
Nucleotide and amino acid allele defintions are available here:
- https://www.ebi.ac.uk/ipd/imgt/hla/download.html
- ftp://ftp.ebi.ac.uk/pub/databases/ipd/imgt/hla/
Convert these definition files to the reference files used by Lilac with the following command:
java -cp lilac.jar com.hartwig.hmftools.lilac.utils.GenerateReferenceSequences \
-resource_dir /path_to_definition_files/ \
-output_dir /output_dir/ \
| Argument | Description |
|---|---|
| sample | Sample ID |
| ref_genome | Reference genome fasta file |
| reference_bam | Sample's germline BAM |
NOTE: Lilac handles BAMs which have been sliced for the HLA gene regions.
If a sample's tumor BAM is provided in place of the reference BAM, then Lilac will determine the allele solution from it instead.
| Argument | Description |
|---|---|
| ref_genome_version | V37 (default), V38 or HG19 (ie 37 with 'chr' prefix) |
| tumor_bam | Sample's tumor BAM |
| rna_bam | Sample's RNA BAM if available |
| gene_copy_number_file | Sample gene copy number file from Purple |
| somatic_variants_file | Sample's somatic variant VCF file, for annotation of HLA gene variants |
| Argument | Default | Description |
|---|---|---|
| min_base_qual | 30 | Min base quality for BAM reads, see documentation for details |
| min_evidence | 2 | Min absolute required fragments for evidence phase |
| min_evidence_factor | 0.00075 | Min relative required fragments for evidence phase, as a fraction of total fragments |
| min_high_qual_evidence_factor | 0.000375 | Minimum relative required high base-quality fragments for evidence phase, as a fraction of total fragments |
| min_fragments_per_allele | 7 | See documentation for details |
| min_fragments_to_remove_single | 40 | See documentation for details |
| top_score_threshold | 5 | Maximum difference in candidate solution score vs top score as a percentage of total fragments |
| log_debug | Off (logs at INFO) | Logs in verbose mode |
| debug_phasing | Off | Logs phasing evidence construction |
| expected_alleles | Not applied | List of alleles separated by ';'. These alleles will have their coverage and ranking reported even if not in the winning solution |
| restricted_alleles | Not applied | List of alleles separated by ';'. Restrict evaluation to only these alleles. |
| threads | 1 | Number of threads to use for complex evaluation |
java -jar lilac.jar \
-sample COLO829T
-ref_genome /path_to_ref_genome_fasta_file/ \
-resource_dir /path_to_lilac_resource_files/ \
-reference_bam /sample_data_path/COLO829R.bam \
-output_dir /output_dir/ \
Or with tumor BAM, somatic variants VCF and Purple gene copy number inputs:
java -jar lilac.jar \
-sample COLO829T
-ref_genome /path_to_ref_genome_fasta_file/ \
-resource_dir /path_to_lilac_resource_files/ \
-reference_bam /sample_data_path/COLO829R.bam \
-tumor_bam /sample_data_path/COLO829T.bam \
-somatic_vcf /sample_data_path/COLO829T.purple.somatic.vcf.gz \
-gene_copy_number /sample_data_path/COLO829T.purple.cnv.gene.tsv \
-output_dir /output_dir/ \
The following files are written:
| Field | Description |
|---|---|
| Allele | Allele ID |
| RefTotal | Total assigned fragments from reference BAM |
| RefUnique | Fragments uniquely assigned to allele |
| RefShared | Fragments assigned to allele and others in this solution |
| RefWild | Fragments matched to a wildcard allele |
| TumorCopyNumber | Copy number from Tumor/Ref fragment ratio and Purple copy number |
| TumorTotal | As above for tumor BAM |
| TumorUnique | As above for tumor BAM |
| TumorShared | As above for tumor BAM |
| TumorWild | As above for tumor BAM |
| RnaTotal | As above for RNA BAM |
| RnaUnique | As above for RNA BAM |
| RnaShared | As above for RNA BAM |
| RnaWild | As above for RNA BAM |
| SomaticMissense | Matched missense variants |
| SomaticNonsenseOrFrameshift | Matched nonsense or frameshift variants |
| SomaticSplice | Matched splice variants |
| SomaticSynonymous | Matched synonymous variants |
| SomaticInframeIndel | Matched inframe indels |
| Field | Description |
|---|---|
| Status | PASS, otherwise 1 or more warnings |
| HlaY | true/false, if exceeds HLA threshold (10% of total fragments) |
| ScoreMargin | Difference in score to second-top solution |
| NextSolutionAlleles | Allele difference in second-top solution |
| MedianBaseQuality | Median base quality across all coding bases from all fragments |
| DiscardedIndels | Discarded fragments due to unknown INDELs |
| DiscardedIndelMaxFrags | Maximum fragments assigned to any particular unknown INDEL |
| DiscardedAlignmentFragments | |
| A_LowCoverageBases | Number of bases with less than 15-depth coverage across all coding bases, also for B and C genes |
| ATypes | Number of HLA-A alleles |
| BTypes | Number of HLA-B alleles |
| CTypes | Number of HLA-C alleles |
| TotalFragments | Total fragments used to fit candidate solutions |
| FittedFragments | Total fragments used in top solution |
| UnmatchedFragments | Fragments fitted to other solutions |
| UninformativeFragments | Fragments covering only homozygous or wildcard regions |
| HlaYFragments | Fragments assigned to HLA-Y |
| PercentUnique | Percent of unique fragments in solution |
| PercentShared | Percent of shared fragments in solution |
| PercentWildcard | Percent of wildcard-allele-only fragments in solution |
| UnusedAminoAcids | Distinct unmatched amino acids |
| UnusedAminoAcidMaxFrags | Maximum fragments assigned to a distinct unmatched amino acids |
| UnusedHaplotypes | Distinct unmatched haplotypes |
| UnusedHaplotypeMaxFrags | Maximum fragments assigned to a distinct unmatched haplotype |
| SomaticVariantsMatched | Somatic variants supported by solution allele |
| SomaticVariantsUnmatched | Somatic variants not supported by solution allele |
Additional output files
| File | Description |
|---|---|
| SAMPLE_ID.lilac.somatic.vcf.gz | Annotation of HLA gene somatic variants if somatic VCF provided |
| SAMPLE_ID.fragments.csv | Read details for all BAM fragments |
| SAMPLE_ID.candidate.coverage.csv | Coverage for all candidate solutions within X% of the top solution's score |
| SAMPLE_ID.candidate.fragments.csv | Allocation of each fragment to one or more solutions and which alleles they support |
| SAMPLE_ID.HLA-A.aminoacids.txt | Fragment support for each amino acid by HLA gene |
| SAMPLE_ID.HLA-A.nucleotides.txt | Fragment support for each nucleotide by HLA gene |
| SAMPLE_ID.candidates.aminoacids.txt | Fragment support for amino acids in the candidate alleles |
| SAMPLE_ID.candidates.nucleotides.txt | Fragment support for nucleotides in the candidate alleles |