PGnome^® - Whole Genome Sequencing

PGnome Diagnostic has a TAT of 5-7 weeks 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.

• 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.

Reports will consist of up to six different sections:

Primary Findings (related to the indication for testing)

• Variants in genes known to be associated with phenotype, including VUS
• Variants in genes possibly associated with phenotype, including VUS

Secondary Findings (if opted in on the requisition form)

• Guideline Recommended Genes: Recent recommendations are that labs performing WES or WGS should report pathogenic variants in selected genes that cause (mostly) dominantly inherited disorders (Version 3.2, Miller et al. 2023. PubMed ID: 37347242). These disorders are treatable and/or preventable. Included on this list are some cancer predisposition conditions, heart conditions associated with sudden death, and conditions that could result in severe health consequences if surgery is performed with certain anesthetics. Only pathogenic and likely pathogenic variants are reported.
• Other Predispositions/Diagnoses: This secondary finding option refers to a very broad range of disorders, including adult-onset neurological conditions such as Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis (ALS), small vessel disease, and renal disease, among others. Some of these disorders are very serious, leading to death. Treatment or prevention will be effective for some of these disorders but not for others. Knowledge of these predispositions may be useful for the patients and their families (Amendola et al. 2015. Genome Res 25(3):305- 315; Dorschner et al. 2013. Am J Hum Genet 93(4):631-640).  If this option is selected, we will report all variants that are likely to result in a Mendelian (single gene) disorder (i.e., one variant in a dominant gene or X-linked gene or two variants in a recessive gene). Many of these conditions have adult onset, and in accordance with current professional guidelines (Borry et al. 2006 Clin Genet 70(5):374-81; Lucassen et al. 2010 British Society for Human Genetics; Fallat et al. 2013 Pediatrics 131(3): 620–2; NSGC Position Statement 2017)  we do not recommend release of information about adult-onset conditions to minors (under the age of 18 years). For minors, we recommend that this testing be postponed until the age of 18 years or that access to this portion of their healthcare records be blocked until they reach 18 years.  Only pathogenic and likely pathogenic variants are reported. Individuals will be screened for expansions in ATXN2, c9orf72, FMR1, PABPN1 and PHOX2B if they opt-in to this category. Variants in the mitochondrial genome will not be reported in this category.
• Carrier Status: Variants in any gene that relate to an autosomal recessive or X-linked recessive disorder in females will be reported if this option is selected (regardless of the incidence of the condition). Such single recessive, pathogenic variants usually don’t appreciably affect a patient’s health, but may be useful in reproductive planning. In accordance with current professional guidelines Borry et al. 2006. Eur J Hum Genet 14(2):133-8; NSGC Position Statement 2012; Ross et al. 2013 Genet Med 15(3):234-245), we do not recommend release of carrier information to minors (under the age of 18 years), we do not recommend release of carrier information to minors (under the age of 18 years). For minors, we recommend that carrier testing be postponed until the age of 18 years or that access to this portion of their healthcare records be blocked until they reach 18 years. Only pathogenic and likely pathogenic variants are reported. Females who opt-in to carrier status findings will also be screened for FMR1 CGG-repeat expansion status. Variants in the mitochondrial genome will not be reported in this category.
• PG Discovery (Candidate Genes, Available for Trios with Parents Only): WGS provides the opportunity to identify rare variants in candidate genes for which there is limited available evidence. Relevant rare homozygous, hemizygous, compound heterozygous, and/or de novo variants are reported. These genes and variants reported within them will be classified as uncertain significance, and the variants will not be confirmed by a second method (usually Sanger sequencing). Any literature, such as limited animal studies, etc., is referenced where available. Further research is required to understand if any human disease association exists. PreventionGenetics may reach out to request consent for submission of these variants to research programs and databases like GeneMatcher (https://genematcher.org/). Genetic variants related to complex disease, and mitochondrial disorders (excluding nuclear genes) will not be reported at this time. Only uncertain variants (VUS) are reported.

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

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

Alfares et al. 2018. PubMed ID: 29565419

Amendola et al. 2015. PubMed ID: 25637381

Borry et al. 2006. PubMed ID: 17026616

Dorschner et al. 2013.. PubMed ID: 24055113

Elliott et al. 2019. PubMed ID: 31172278

Fallat et al. 2013 Pediatrics 131(3): 620–2; NSGC Position Statement 2017

Farnaes et al. 2018. PubMed ID: 29644095

Farwell et al. 2015. PubMed ID: 25356970

Lindstad 2022.

Lionel et al. 2018. PubMed ID: 28771251

Lucassen et al. 2010 British Society for Human Genetics

Miller D. et al. 2023. Genetics in Medicine : Official Journal of the American College of Medical Genetics. PubMed ID: 37347242

NSGC Position Statement 2012

Ross et al. 2013 Genet Med 15(3):234-245

Stark et al. 2017. PubMed ID: 28125081

Vissers et al. 2017. PubMed ID: 28333917