PGnomeTM - Whole Genome Sequencing

Test Requisition Form

PGnomeTM Diagnostic

Name Test Code Description CPT Code(s) Price
Family - Trio 8001 WGS of patient + 2 additional family members 81425, 81426(x2) $4,890

If report is needed for any additional family members, add $490 per family member.

Family - Duo 8000 WGS of patient + 1 additional family member 81425, 81426 $3,790

If report is needed for any additional family members, add $490 per family member.

Patient Plus 7001 WGS of patient + targeted variant testing of parents (both parents required) 81425 $2,590
Patient Only 7000 WGS of patient 81425 $2,490

Sequencing cost to additional family members beyond trio: $1,290 (no report); additional CPT Code 81426.

If report is needed for any additional family members, add $490 per family member.

What is PGnome?

PGnome is PreventionGenetics' whole genome sequencing (WGS) test. PGnome is the ultimate germline DNA test because it covers the entire genome. Although initially the primary application of WGS will be diagnosis, there are other, very powerful applications as shown in the following list.

Primary Applications of WGS

  • Diagnosis
  • Assessment of Disease Risk and Prevention
  • Reproductive Planning
  • Pharmacogenetics
  • Research

The clinical utility of genome and exome sequencing for diagnosis of disease is now abundantly clear (see for example Lunke et al. 2020. PubMed ID: 32573669; Clark et al. 2019. PubMed ID: 31019026; Elliott et al. 2019. PubMed ID: 31172278; Mestek-Boukhibar et al. 2018. PubMed ID: 30049826; Meng et al. 2017. PubMed ID: 28973083; Stark et al. 2017. PubMed ID: 28125081) Genome/exome sequencing is superior to sequencing of single genes or smaller gene panels because of genetic heterogeneity, the continuing discovery of new disease genes, dual diagnoses (which are surprisingly common; see for example Karaca et al. 2018. PubMed ID: 29790871), and the general difficulty of identifying the correct genes using clinical features alone. Genome/exome sequencing also often reduces the time to diagnosis, limiting the diagnostic odyssey and lowering the cost to patients.

Genome sequencing is superior to exome sequencing because it covers portions of the genome like deep intronic regions that are not covered by exome sequencing and because it yields better detection of Structural Variants (defined as Copy Number Variants (CNVs) plus inversions, and translocations). In addition, genome sequencing provides more accurate analysis of tandem repeats and paralogous regions, and is essential for application of polygenic risk algorithms. The great majority of variants used by polygenic risk algorithms are not located in coding regions and are therefore missed entirely by exome sequencing. A patient receiving exome sequencing today will likely have to pay again in future for genome sequencing.

The diagnostic yield of WGS varies considerably depending upon the disorder(s) and the groups of patients involved. Yields as high as 60-70% have been reported (Elliott et al. 2019. PubMed ID: 31172278; Stark et al. 2017. PubMed ID: 28125081). However, based on our own experience and other reports from the literature, yields in the range of 30% seem overall more realistic (Farnaes et al. 2018. PubMed ID: 29644095; Lionel et al. 2018. PubMed ID: 28771251; Vissers et al. 2017. PubMed ID: 28333917). Further, based on reports from the literature as well as our own internal data, trio testing (a proband along with both parents) provides higher diagnostic yields than testing just the proband (Farwell et al. 2015. PubMed ID: 25356970). Trio testing is the gold standard as it permits the identification of de novo variants as well as the phase of two different variants in recessive genes immediately upon data review.

PGnome - Diagnostic is ideal for individuals with:

  • Disorders with significant genetic heterogeneity
  • Global developmental delay/intellectual disability, with or without dysmorphic features
  • Dysmorphic features, multiple congenital anomalies, or birth defects

To order PGnome - Diagnostic:

PGnome Diagnostic Test Requisition Form & Provider Statement

TURN AROUND TIME (TAT)

PGnome Diagnostic has a TAT of 28 calendar days on average.

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.

ORDERING / SPECIMENS

Our PGnome Diagnostic offers the traditional options of Patient Only testing or Family testing (e.g., Duo, Trio, etc.), but also offers our Patient Plus testing option. For Patient Plus, we require sending in both biological parents along with the patient's specimen. However, genome sequencing is performed only on the patient's specimen, and depending on variants identified and to be reported in the proband, parental specimens are then used for targeted testing to determine the phase of variants or to determine if a variant occurs de novo. Parents are tested for all sequencing variants included in the proband's report, except for CNVs. For the highest diagnostic rate, Family - Trio testing is recommended.

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.

 

TEST METHODS

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: >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.  All reported variants are confirmed by a second method (usually Sanger sequencing).

Structural variants (SVs) are also detected from NGS data. Our SV calling algorithm (Parliament 2) utilizes read depth, SNP information, split reads, and reads which map to two different sites in the genome.  SV detection via analysis of WGS data is an area of very active research and development.  Improvements will come rapidly.  At this time, we are limiting our SV detection to deletions larger than 1 kb in size, duplications (no size limit) and inversions.  For these SVs, our overall sensitivity during validation was 86%. Sensitivity for ≥ 1 kb deletions alone is > 95%.

REPORTING

Reports will consist of up to six different sections:

Primary Findings (related to the indication for testing) (Only pathogenic, likely pathogenic and uncertain variants are reported.)

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

Secondary Findings (only if specifically requested on test requisition form)

  • Recommended 59 Genes: Medically actionable variants from the ACMG recommended list of genes (Kalia et al. 2016).  Only pathogenic and likely pathogenic variants are reported.
  • Other Predispositions/Diagnoses: Variants in genes not associated with phenotype but result in a Mendelian disorder.  Only pathogenic and likely pathogenic variants are reported.
  • Carrier Status: Carrier status for variants that are causative for recessive disease.  Only pathogenic and likely pathogenic variants are reported.
  • PG Discovery (available for trios only): Relevant rare variants in candidate genes for which there is limited available evidence.  Only uncertain variants are reported.

All differences from the reference sequences (sequence variants) 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). Benign and likely benign variants are not reported.  A full list of all sequence variants will be provided to the ordering physician upon request.

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

LIMITATIONS AND OTHER TEST NOTES

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 this specific disorder are accumulated.

Sequencing: When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

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.

Our detailed variant analysis and interpretation is focused on the coding exons and immediate flanking non-coding DNA (± 10 bp).  Although the millions of variants detected in other parts of the genome are used to assist with SV detection and other applications, we do not at this time attempt to interpret every variant outside of coding and immediate flanking regions.

In many 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.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.

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.

Structural Variants (SVs): The PGnome test uses a program called Parliament2 to detect SVs.  Parliament2 combines output from five SV calling programs (Breakdancer, CNVnator, Delly, Lumpy and Manta). At this time, we are limiting our SV detection to deletions larger than 1 kb in size, duplications (no size limit), and inversions. For these SVs, our overall sensitivity during validation was 86%. Sensitivity for ≥ 1 kb deletions alone is > 95%. Some SVs will not be detected due to paralogy (e.g., pseudogenes, segmental duplications), sequence properties, and size.  Sensitivity for detection of insertions (as opposed to duplications) with Parliament2 is low (~20%).  At this time, we are not reporting translocations. Our ability to detect SVs due to somatic mosaicism is limited.

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. Results of PGnome testing can be used for both diagnostic and scientific research purposes.

CONTACTS

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: James Weber, PhD - jim.weber@preventiongenetics.com

REFERENCES

Clark et al. 2019. PubMed ID: 31019026

Elliott et al. 2019. PubMed ID: 31172278

Farnaes et al. 2018. PubMed ID: 29644095

Kalia et al. 2016. PubMed ID: 27854360

Karaca et al. 2018. PubMed ID: 29790871

Lionel et al. 2018. PubMed ID: 28771251

Lunke et al. 2020. PubMed ID: 32573669

Meng et al. 2017. PubMed ID: 28973083

Mestek-Boukhibar et al. 2018. PubMed ID: 30049826

Richards et al. 2015. PubMed ID: 25741868

Stark et al. 2017. PubMed ID: 28125081

Vissers et al. 2017. PubMed ID: 28333917