PGnome^® - Whole Genome Sequencing

Nearly all Rapid PGnome tests have a TAT of 2 weeks on average.

We recommend that providers choose expedited shipping to decrease the time samples spend in transit to PreventionGenetics.

Inclusion of detailed clinical notes/completion of the clinical data checklist and a pedigree are required. The ability to select variants that may be involved with the patient’s health problem directly correlates with the quality of clinical information provided.

• Singleton – WGS on proband sample only.
• Duo – WGS on proband and comparator sample.
• Trio – WGS on proband and two comparator samples. Note: sensitivity is highest when biological parent samples are used as comparators.

If ordering a Duo or Trio test, the proband and all comparator samples are required to initiate testing. If we do not receive all required samples for the test ordered within 21 days, we will convert the order to the most effective testing strategy with the samples available. Prior authorization and/or billing in place may be impacted by a change in test code.

Comparator samples will only be interrogated for variants found in the proband, and the presence or absence of those findings in the comparator will be included on the proband report.

Specimen Requirements and Shipping Details

Note that saliva and buccal specimens are not accepted for WGS. DNA from saliva invariably includes microbial and food DNA which interfere with WGS.

Only primary findings will be reported for Rapid PGnome. Reports will consist of up to two sections:

• Variants in genes known to be associated with phenotype
• Variants in genes possibly associated with phenotype

A preliminary report prior to confirmation may be issued in cases with a clear positive finding.

Secondary findings are not reported.

Benign and likely benign variants are not reported.

Raw sequence data will be provided to the ordering physician upon request.

Nomenclature for sequence variants comes from Human Genome Variation Society (HGVS) (https://www.hgvs.org).

Sequencing: PGnome uses Illumina short-read next generation sequencing (NGS) technologies. As required, genomic DNA is extracted from patient specimens. Patient DNA is sheared, adaptors are ligated to the fragment ends, and the fragments are sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads. The following quality control metrics are generally achieved for the nuclear genome: >98% of targeted bases are covered at >15x, >96% of targeted bases are covered at >20x.  The minimum acceptable average read depth is 35x. Data analysis and interpretation is performed by the internally developed Infinity pipeline. Variant calls are made by the GATK Haplotype caller and annotated using in house software and Jannovar.

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (https://www.hgvs.org).  All differences from the reference sequences are assigned to one of five interpretation categories (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign and Benign) per ACMG Guidelines (Richards et al. 2015. PubMed ID: 25741868).  Benign and likely benign variants are not reported.

Structural Variants: Three SV calling algorithms are employed (Lumpy, CNVnator, and Manta) that utilize read depth, SNP information, split reads, and reads which map to two different sites in the genome to detect deletions, duplications, insertions and inversions. The overall sensitivity for deletions, duplications, and inversions is 96%. Sensitivity for detection of insertions (as opposed to duplications) is currently low (~20%). At this time, balanced translocations are not reported.  The ability to detect SVs due to somatic mosaicism is limited.

Mitochondrial Genome: For the mitochondrial genome screen, the following quality control metrics are generally achieved: an average read depth of >3,000x and a minimum acceptable read depth of 500x. Variant calls for the mitochondrial genome are made using the Mutserve pipeline and annotated using Alamut batch.  At this time, structural variants within the mitochondrial genome are not reported.

Mitochondrial analysis is only available for the proband at this time.

Repeat Expansions: For the repeat expansion screen, individuals with phenotypes and/or family history consistent with ATXN2, c9orf72,  PABPN1, PHOX2B, and/or FMR1-related disease or who opt-in for ‘Other Predispositions/Diagnoses’ secondary findings will be evaluated for expansions in these genes. In addition, female patients that opt-in for Carrier Status findings will be screened for expansions in FMR1. Our genome test screens for expanded allele repeat sizes in ATXN2, c9orf72, FMR1, PABPN1, and PHOX2B with nearly 100% analytical specificity and sensitivity. Any potential expansions detected by the screening test will be confirmed with the appropriate confirmatory clinical repeat expansion test. Only those expansions that are confirmed will be reported. 

Limitations of NGS: Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and genetic disorders improves.

Sequencing: This test will not cover 100% of the genome. Parts of the genome cannot be readily sequenced with current technology such as some tandem repeats, paralogous genes and other repeat sequences. Therefore, a small fraction of sequence variants relevant to the patient's health will not be detected.

When Next Generation Sequencing (NGS) or Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not capture or amplify, due for example to a large deletion or insertion.

Detailed variant analysis and interpretation is focused on the coding exons and immediate flanking non-coding DNA (± 10 bp). We do not attempt to interpret every variant outside of coding and immediate flanking regions. When warranted by sequence results (for example a single pathogenic variant in a recessive gene), rare variants within selected genic regions will be investigated.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative variants for recessive disorders, we cannot be certain that the variants are on different alleles.

The ability to detect low-level mosaicism of variants is limited.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct. Genome build hg19, GRCh37 (Feb2009) is used as our reference in nearly all cases.

Mitochondrial variants: We analyze nearly the entire genome, although certain regions (<200 bp) are excluded from analysis largely due to the presence of tandem repeats in non-coding regions. This test is currently not validated to detect large deletions, duplications, or complex rearrangements of the mitochondrial genome, and our sensitivity for smaller insertion/deletion events is also limited. Based on downsampling experiments, at our minimum coverage depth of 500x, sensitivity for detection of single nucleotide variants at ≤4% heteroplasmy was 99.85%, with a positive predictive value of 100%. At ≤10% heteroplasmy, sensitivity for single nucleotide variants was 100.0% with a positive predictive value of 100.0%. Therefore, although sensitivity for detection of low-level (4-10%) heteroplasmic single nucleotide variants is expected to be high based on validation studies, we cannot guarantee that these low-level heteroplasmic variants will always be identified due to paralogy with the nuclear genome.

Structural Variants (SVs): Sensitivity for detection of insertions (as opposed to duplications) is currently low (~20%). At this time, we are not reporting translocations. Our ability to detect SVs due to somatic mosaicism is limited. On occasion, it will not be technically possible to confirm a smaller SV called by NGS. In these instances, the SV will not be included on the report. 

Repeat Expansion Screening: Tests that meet our eligibility criteria for repeat expansion screening (described above in Test Methods) must have locus coverage of at least 20x for data to be considered reliable. This screen may not detect low-level mosaic expansions. Any potential expansions will be confirmed with an appropriate confirmatory repeat expansion test. Only results from the confirmatory repeat expansion test (including repeat count and methylation status, if applicable) will be included in the final report.

General: We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the specimen arrives at PreventionGenetics.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

Genetic Counselors: GC Team - support@preventiongenetics.com

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