PGnome® - Whole Genome Sequencing
PGnome® Health Screen
What is PGnome?
PGnome is PreventionGenetics' whole genome sequencing (WGS) test. PGnome is the ultimate germline DNA test because it covers the entire genome. The PGnome Health Screen is intended for patients who are basically healthy but who want to learn their carrier status for recessive disease, their susceptibility to adult onset disorders, or both. The primary applications of WGS are shown in the following list.
Primary Applications of WGS
- Assessment of Disease Risk and Prevention
- Reproductive Planning
Although we sequence all human genes for the PGnome Health Screen, we analyze and report sequence variants and copy number variants (CNVs) only in genes that have been proven with high confidence to be involved in Mendelian (also called single gene) disorders (MacArthur et al. 2014. PubMed ID: 24759409). Our list of "clinically-relevant" genes currently includes about 5500 genes and is updated quarterly. We do not report variants in genes that for technical reasons cannot be accurately sequenced (primarily due to the presence of pseudogenes).
In addition, although we identify and interpret all sequence variants (differences between the patient's sequence and the reference sequence (build hg19)), we report only pathogenic and likely pathogenic variants (Richards et al. 2015. PubMed ID: 25741868).
We have found through our exome sequencing at PreventionGenetics that the average person is a recessive disease carrier for less than five pathogenic or likely pathogenic variants. Note, however, that the average person also carries over 100 variants of uncertain significance and millions of benign variants.
For this test, patients also have the option of receiving results of pathogenic and likely pathogenic variants in genes that predispose to or confirm a diagnosis of adult onset disorders such as cancer and heart disease (Kalia et al. 2016. PubMed ID: 27854360).
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.
TURN AROUND TIME (TAT)
PGnome Health Screen has a TAT of 28 calendar days on average.
ORDERING / SPECIMENS
Singleton Pricing (sequencing and report): $2,290
Couple Pricing (sequencing and report for each person): $3,990
Test Code: #9000 (singleton); #9001 for couples
CPT Code: 81425 (x2 if couple)
Note that saliva and buccal specimens are not accepted for WGS. DNA from saliva invariably includes microbial and food DNA which interfere with WGS.
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 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%.
Reports will consist of up to three different sections:
- Variants that are causative for recessive disease
- Medically actionable variants from the ACMG recommended list of genes, if requested (Kalia et al. 2016. PubMed ID: 27854360)
- Variants in other genes that result in a dominant Mendelian disorder, if requested
Only Pathogenic and Likely Pathogenic variants are reported for PGnome Health Screen.
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 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): Calling of SVs from short read sequence data is challenging and a very active area of research and development. Improvements will come relatively quickly. However, 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) is currently 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.
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.
Genetic Counselors: GC Team - firstname.lastname@example.org
Geneticist: James Weber, PhD - email@example.com
Kalia et al. 2016. PubMed ID: 27854360
MacArthur et al. 2014. PubMed ID: 24759409
Richards et al. 2015. PubMed ID: 25741868