snv-indels module

The snv-indels module is responsible for aligning the reads to the reference, and calling variants. The bam and count files produced by this module are used in the fusion and gene expression modules.

Tools

This module uses STAR to align the reads to the reference using two-pass mode. VarDict is used to call variants, which are annotated using VEP. Variants are filtered based on the criteria defined in filter_criteria, and annotated based on annotation_criteria.

The variants annotated by VEP are then filtered based on a number of different criteria:

Variants that are present on the blacklist are excluded.
Only variants that match at least one criteria in filter_criteria are included.
Variant that have a population frequency of more than 1% in the gnomADe population are excluded.

Picard is used to generate various alignment statistics.

Input

The input for this module is a single pair of FastQ files per sample, specified in a PEP configuration file, as is shown below.

Example input for the snv-indels module
sample_name	R1	R2	strandedness
MO1-RNAseq-1-16714	test/data/fastq/NOMO1-RNAseq-1-16714_R1.fastq.gz	test/data/fastq/NOMO1-RNAseq-1-16714_R2.fastq.gz	reverse

Output

The output of this module are a JSON file with an overview of the most important results, as well as a number of other output files:

A .bam and .bai per sample, which contain the aligned reads.
The filtered VEP output file (filter_vep), which contains the final set of filtered and annotated variants.
The counts file produced by STAR, which contains the coverage per gene.

Configuration

You can automatically generate a configuration for the fusion module using the utilities/create-config.py script.

Example

{
  "genome_fasta": "test/data/reference/hamlet-ref.fa",
  "genome_fai": "test/data/reference/hamlet-ref.fa.fai",
  "genome_dict": "test/data/reference/hamlet-ref.dict",
  "star_index": "test/data/reference/hamlet-star",
  "ref_id_mapping": "test/data/reference/id_mappings.tsv",
  "filter_criteria": "test/data/config/filter_criteria.tsv",
  "annotation_criteria": "test/data/config/annotation_criteria.tsv",
  "rrna_refflat": "test/data/reference/ucsc_rrna.refFlat",
  "gtf": "test/data/reference/hamlet-ref.gtf",
  "annotation_refflat": "test/data/reference/hamlet-ref.refFlat"
}

Note that the vep-cache entry is missing for this example file, which means that VEP will be run with only the fasta and gtf files as input. For the best performance, please specify a vep-cache folder as well.

Configuration options

Configuration options
Option	Description	Required
genome_fasta	Reference genome, in FASTA format	yes
genome_fai	.fai index file for the reference fasta	yes
genome_dict	.dict index file for the reference fasta	yes
star_index	STAR index database	yes
ref_id_mapping	File of transcripts of interest	yes
filter_criteria	Criteria file to filter variants	yes
annotation_criteria	Criteria file to annotate variants	yes
rrna_refflat	File of rRNA transcripts	yes
gtf	GTF file with transcripts, used by STAR	yes
annotation_refflat	File used to determine exon coverage	yes
blacklist	File of blacklisted variants	no
vep-cache	Folder containing the VEP cache	no
variant_allele_frequency	Minimum variant allele frequency in the sample to call a variant (default=0.05)	no

Filter and annotation criteria

HAMLET include the ability to specify separate filter criteria for every transcript, based on the position and the VEP consequence of the variant. The criteria are used both the filter which variants will be part of the output (filter_criteria), and also annotate the identified variants (annotation_criteria).

The required columns are transcript_id, consequence, start and end. For annotation variants, the annotation column is used. Every column except for transcript_id can be empty.

Example `filter_criteria` file, from the HAMLET tests
transcript_id	consequence	start	end
ENST00000361851.1	stop_gained
ENST00000361851.1	frameshift_variant
ENST00000361851.1	stop_lost
ENST00000361851.1	start_lost
ENST00000361851.1	inframe_insertion
ENST00000361851.1	inframe_deletion
ENST00000361851.1	protein_altering_variant
ENST00000361851.1	missense_variant
ENST00000361899.1	stop_gained
ENST00000361899.1	frameshift_variant
ENST00000361899.1	stop_lost
ENST00000361899.1	start_lost
ENST00000361899.1	inframe_insertion
ENST00000361899.1	inframe_deletion
ENST00000361899.1	protein_altering_variant
ENST00000361899.1	missense_variant
ENST00000361789.1	stop_gained
ENST00000361789.1	frameshift_variant
ENST00000361789.1	stop_lost
ENST00000361789.1	start_lost
ENST00000361789.1	inframe_insertion
ENST00000361789.1	inframe_deletion
ENST00000361789.1	protein_altering_variant
ENST00000361789.1	missense_variant
ENST00000241453.1	inframe_insertion

Example `annotation_criteria` file, from the HAMLET tests
transcript_id	consequence	start	end	annotation
ENST00000241453.1	inframe_insertion	1790	1801	Hotspot
ENST00000361899.1	missense_variant	334	334	Hotspot
ENST00000361851.1	missense_variant	25		Hotspot