Systematic Noise Filtering
When Local Analysis Software is used in the Tumor-Normal or Tumor-Only somatic mode, a BED file with site-specific noise level can be specified for the purpose of filtering out sequencing / systematic noise. The site-specific noise level is used to calculate an AQ score, similar to the Phred-scale. If the AQ score is smaller than the defined threshold, the variant is filtered as systematic noise. The systematic noise BED file is built using VCFs that were generated by the Local Analysis Software Somatic Pipeline when run on normal samples that do not necessarily match to the subject the tumor sample was taken from. The file might contain several dozen samples, ideally normal samples collected on the same library prep kit and sequencing system, so that if there are systematic errors that occur during library prep or sequencing, they are captured in the systematic noise BED file.
The following are the available systematic noise command-line options
| • | --vc-systematic-noise—Specifies a systematic noise BED file. If a somatic variant does not pass the AQ threshold, the variant is marked as systematic_noise in the FILTER column of the output VCF. |
| • | --vc-systematic-noise-filter-threshold—Set the AQ threshold. By default the threshold value for tumor-normal is 10 and 60 for tumor-only. |
Several prebuilt systematic noise files for WGS and WES can be downloaded. For best performance normal samples collected on the same library prep kit and sequencing system should be used to build the systematic noise BED file.
Generate Systematic Noise BED File
You can generate systematic noise BED files from normal samples collected using library prep, sequencing system, and panels. If using panel sequencing, 50 samples are recommended.
To generate a BED file, do as follows.
| 1. | Run DRAGEN somatic tumor-only on normal samples with --vc-systematic-noise set to true to generate VCF output per normal sample. |
| 2. | Build the BED file using the VCFs and the following options. |
| • | --vc-systematic-noise-raw-input-list—List of input VCFs. Enter one VCF per line. |
| • | --vc-systematic-noise-germline-vaf-threshold—Minimum VAF to remove potential germlines from systematic noise file building. Variants with a VAF greater than the threshold are not considered systematic noise. The default is none. |
If using small panels, the recommended threshold is 0.3.
| • | --vc-systematic-noise-use-germline-tag—Use DRAGEN internal germline tagging to remove potential germlines. Mutually exclusive with --vc-systematic-noise-germline-vaf-threshold. The default is false. |
If using WGS or WES, the recommended setting is true.
| • | --vc-systematic-noise-method—Method to calculate the systematic noise level (noise allele frequency) across samples. Enter mean to calculate the average noise allele frequency, max to calculate the maximum, or aggregate to calculate total alleles / total depth per locus across samples. The default is mean. |
If using WGS the recommended setting is max. If using WES, the recommended setting is aggregate. If using small panels for higher sensitivity, the recommended setting is mean or aggregate.
You can also build systematic noise BED files in the cloud using the BaseSpace Sequence Hub DRAGEN CNV Baseline Builder App.
Prebuilt Systematic Noise BED Files
The following prebuilt systematic noise files for WGS and WES are available for download on the Illumina DRAGEN BIo-IT Platform Support Site page.
|
Pre-built Systematic Noise File |
Comment |
Number of Normal Samples |
|---|---|---|
|
WGS_hg38_v1.0_systematic_noise.bed.gz |
WGS hg38 |
28 Samples, mixture of PCRFree NovaSeq, PCRFree HiSeqX, TruSeq Nano HiSeqX |
|
WGS_hs37d5_v1.0_systematic_noise.bed.gz |
WGS hs37d5 |
31 Samples, mixture of PCRFree NovaSeq, PCRFree HiSeqX, TruSeq Nano HiSeqX |
|
WGS_hg19_v1.0_systematic_noise.bed.gz |
WGS hg19 |
31 Samples, mixture of PCRFree NovaSeq, PCRFree HiSeqX, TruSeq Nano HiSeqX |
|
WES_Nextera_IDT_hg38_v1.0_systematic_noise.bed.gz |
Nextera library prep; IDT exome; hg38 |
47 Samples |
|
WES_Nextera_IDT_hs37d5_v1.0_systematic_noise.bed.gz |
Nextera library prep; IDT exome; hs37d5 |
47 Samples |
|
WES_Nextera_IDT_hg19_v1.0_systematic_noise.bed.gz |
Nextera library prep; IDT exome; hg19 |
47 Samples |
|
WES_TruSeq_IDT_hg38_v1.0_systematic_noise.bed.gz |
TruSeq library prep; IDT exome; hg38 |
53 Samples |
|
WES_TruSeq_IDT_hs37d5_v1.0_systematic_noise.bed.gz |
TruSeq library prep; IDT exome; hs37d5 |
53 Samples |
|
WES_TruSeq_IDT_hg19_v1.0_systematic_noise.bed.gz |
TruSeq library prep; IDT exome; hg19 |
53 Samples |