PGxome® - Whole Exome Sequencing

PGxome® Diagnostic
Requisition Form

Name Test Code Description CPT Code(s) Price
Family - Trio 5300 WES of patient + 2 additional family members 81415, 81416(x2) $2,490

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

Patient Plus 5005 WES of patient + targeted variant testing of parents (both parents required) 81415 $1,990
Family - Duo 5200 WES of patient + 1 additional family member 81415, 81416 $2,290

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

Patient Only 5000 WES of patient 81415 $1,890

Sequencing cost to additional family members beyond trio: $390 (no report); additional CPT Code 81416

PGxome - Diagnostic is ideal for individuals with:

  • Unresolved genetic testing such as normal karyotyping or microarray analysis, and negative single gene or gene panel sequencing results
  • Disorders with significant genetic heterogeneity
  • Global developmental delay/intellectual disability, with or without dysmorphic features
  • Dysmorphic features, multiple congenital anomalies, or birth defects

What is PGxome Diagnostic?

PGxome is PreventionGenetics' whole exome sequencing (WES) test. The PGxome assesses almost all genes from the human genome including coding regions and adjacent introns. This test is intended for health care providers who are looking for a genetic diagnosis when the clinical phenotype is unclear and/or previous test results have been uninformative. This is important as more than 50% of patients with genetic diseases are not given a specific diagnosis even after repeat clinical examinations and tests (Shashi et al. 2014). The standard clinical practice often involves examinations for specific phenotypes, imaging, biochemical testing for inborn errors of metabolism, genomic tests such as karyotyping or microarrays, and single gene or panel tests (Iglesias et al. 2014). However, patients remain without a genetic diagnosis, and patients and health care providers are caught in a long term diagnosis search, known as a diagnostic odyssey. This can lead to failures in identifying potential treatments, and unknown recurrence and prognosis risks (Yang et al. 2013).

Reported diagnostic rates from commercial and academic laboratories have found that WES assays have a ~20-40% positive diagnostic rate, with higher rates being reported from trio analysis (i.e. proband and parents) compared to singleton analysis (Atwal et al. 2014; Iglesias et al. 2014; Farwell et al. 2015). Notably, ~5-7% of individuals who have WES have had dual diagnoses (i.e. two non-overlapping clinical presentations) (Yang et al. 2014; Farwell et al. 2015; Posey et al. 2016). The inclusion of copy number variant (CNV) calling should increase diagnostic rates. One study reported that 30% of genetics diagnoses have only been recently resolved due to new literature reports, highlighting the fast pace of gene-disease discovery and the need of genetic testing laboratories to be current of the medical literature (Yang et al. 2014). The use of a whole exome sequencing test may aid in altering clinical management, predict recurrence and prognosis risks, and reduce costs of additional testing, and may offer advantages over traditional molecular tests in certain patients (Valencia et al. 2015).

PGxome - Diagnostic is ideal for individuals with:

  • Unresolved genetic testing such as normal karyotyping or microarray analysis, and negative single gene or gene panel sequencing results
  • Disorders with significant genetic heterogeneity
  • Global developmental delay/intellectual disability, with or without dysmorphic features
  • Dysmorphic features, multiple congenital anomalies, or birth defects

TURN AROUND TIME (TAT)

PGxome Diagnostic has a TAT of 30 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 PGxome 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, exome sequencing is performed only on the patient’s specimen, and depending on variants identified specifically related to the patient’s clinical presentation, parental specimens are then used for targeted testing to determine the phase of variants or to determine if a variant occurs de novo. For the highest diagnostic rate, Family - Trio testing is recommended.

Specimen Requirements and Shipping Details

Specimen TypeSpecimen Requirements
Blood

Draw blood in EDTA (purple-top) or ACD (yellow-top) tubes.

  • Infants <2 years old: 1mL-2mL blood in EDTA or ACD tubes
  • Adults and children >2yrs: 3mL-5mL blood in EDTA or ACD tubes
DNASend in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 ng/µl.
Tissue (Fresh frozen)Fresh frozen tissue accepted. Please contact us for details. Fixed, paraffin embedded samples are not accepted.
Cultured CellsShip at least two T25 flasks of confluent cells.

Storage and shipping

T25 flasks need to be shipped in appropriate complete culture medium filled to the brim. Ship T25 flasks at room temperature in an insulated container once 90% confluency is reached and by overnight delivery.

Specimen TypeSpecimen Requirements
SalivaIsohelix™ Saliva Collection kit used according to manufacturer instructions.

TEST METHODS

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. 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. Common benign, likely benign, and low quality variants are filtered from analysis.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All reported CNVs are confirmed using another technology such as aCGH, MLPA, or PCR.

REPORTING

Reports will consist of five different sections:

  1. Variants in genes known to be associated with phenotype
  2. Variants in genes possibly associated with phenotype
  3. Medically actionable variants from the ACMG recommended list of genes (if requested) (Kalia et al. 2016)
  4. Variants in genes not associated with phenotype but result in a Mendelian disorder (if requested)
  5. Carrier status for variants that are causative for recessive disease (if requested)

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). Only relevant Pathogenic, Likely Pathogenic, and Uncertain variants are reported. Likely benign and benign variants are not included in the reports. 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.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants relevant to the patient's health will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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

Copy Number Variant Analysis: The PGxome test detects most deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

The sensitivity of this test is dependent on DNA quality.

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 PGxome testing can be used for both diagnostic and scientific research purposes.

CONTACTS

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Jerry Machado, PhD, DABMG, FCCMG - jerry.machado@preventiongenetics.com

REFERENCES

Atwal P.S. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 717-9. PubMed ID: 24525916

Caudle et al. 2016. Genetics in Medicine. PubMed ID: 27441996

Farwell K.D. et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 578-86. PubMed ID: 25356970

Iglesias A. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 922-31. PubMed ID: 24901346

Kalia S.S. et al. 2016. Genetics in Medicine: Official Journal of the American College of Medical Genetics. Advance online publication. doi:10.1038/gim.2016.190. PubMed ID: 27854360

Posey et al. 2016. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 18: 678-85. PubMed ID: 26633545

Richards S et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 405-24. PubMed ID: 25741868

Shashi V. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 176-82. PubMed ID: 23928913

Valencia C.A. et al. 2015. Frontiers in Pediatrics. 3: 67. PubMed ID: 26284228

Yang Y. et al. 2013. The New England Journal of Medicine. 369: 1502-11. PubMed ID: 24088041

Yang Y. et al. 2014. JAMA. 312: 1870-9. PubMed ID: 25326635

PGxome® Health Screen (Test Code #4000)
Requisition Form

Name Test Code Description CPT Code(s) Price
Singleton Pricing 4000 Sequencing and report 81415 $1,490
Couple Pricing 4200 Sequencing and report for each person 81415 (x2) $2,790

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

Please order through the
Requisition Form

What is PGxome Health Screen?

PGxome is PreventionGenetics' whole exome sequencing (WES) assay. The PGxome assesses almost all genes from the human genome including coding regions and adjacent introns. The PGxome Health Screen is intended for patients who are basically healthy, but who want to learn their carrier status for recessive disease and/or their susceptibility to adult onset disorders, and/or their response to certain prescription medications.

Although we sequence nearly all human genes, 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). Our list of "clinically-relevant" genes currently includes about 4800 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).

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 approximately 75 variants of Uncertain significance and thousands 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).

TURN AROUND TIME (TAT)

PGxome Health Screen has a TAT of 30 calendar days on average.

ORDERING / SPECIMENS

Singleton Pricing (sequencing and report): $1,490

Couple Pricing (sequencing and report for each person): $2,790

PGxome Health Screen Test Requisition Form and Healthcare Provider Statement (Required)

Clinical History: Clinical notes and pedigree/family history information should be included when appropriate

Test Code: #4000

CPT Code: 81415 (x2 if couple)

Specimen Requirements and Shipping Details

Specimen TypeSpecimen Requirements
BloodDraw 3mL-5mL blood in EDTA (purple-top) or ACD (yellow-top) tubes.
DNASend in screw cap tube at least 5µg-10µg of purified DNA at a concentration of at least 20 ng/µl.
Tissue (Fresh frozen)Fresh frozen tissue accepted. Please contact us for details. Fixed, paraffin embedded samples are not accepted.
Cultured CellsShip at least two T25 flasks of confluent cells.

Storage and Shipping

T25 flasks need to be shipped in appropriate complete culture medium filled to the brim. Ship T25 flasks at room temperature in an insulated container once 90% confluency is reached and by overnight delivery.

Specimen TypeSpecimen Requirements
SalivaIsohelix™ Saliva Collection kit used according to manufacturer instructions.

TEST METHODS

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. 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. Common benign, likely benign, and low quality variants are filtered from analysis.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All reported CNVs are confirmed using another technology such as aCGH, MLPA, or PCR.

REPORTING

Reports will consist of three different sections:

  1. Variants that are causative for recessive disease
  2. Medically actionable variants from the ACMG recommended list of genes (if requested) (Kalia et al. 2016)
  3. Variants in other genes that result in a dominant Mendelian disorder (if requested)

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). All sequence variants in appropriate gene regions will be detected and interpreted, but only Pathogenic and Likely Pathogenic variants will be included in the test report.

Likely benign and benign variants are not included in the reports. However, 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.

Due to technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of causative sequence variants will not be detected. For those planning reproduction, we cannot guarantee a child free of genetic disorders.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non- coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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

Copy Number Variant Analysis: The PGxome test detects most deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

The sensitivity of this test is dependent on DNA quality.

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 PGxome testing can be used for both diagnostic and scientific research purposes.

CONTACTS

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Jerry Machado, PhD, DABMG, FCCMG - jerry.machado@preventiongenetics.com

REFERENCES

Caudle et al. 2016. Genetics in Medicine. PubMed ID: 27441996

Kalia S.S. et al. 2016. Genetics in Medicine: Official Journal of the American College of Medical Genetics. Advance online publication. doi:10.1038/gim.2016.190. PubMed ID: 27854360

MacArthur D.G. et al. 2014. Nature. 508: 469-76. PubMed ID: 24759409

Richards S. et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 405-24. PubMed ID: 25741868

PGxome® RAPID
Requisition Form

Name Test Code Description CPT Code(s) Price
Family - Trio 13003 WES of patient + 2 additional family members 81415, 81416(x2) $2,890

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

Patient Plus 13000 WES of patient + targeted variant testing of parents (both parents required) 81415 $2,390
Family - Duo 13002 WES of patient + 1 additional family member 81415, 81416 $2,690

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

Patient Only 13001 WES of patient 81415 $2,290

Sequencing cost to additional family members beyond trio: $390 (no report); additional CPT Code 81416

Rapid PGxome - Diagnostic is ideal for:

  • Seriously ill patients with an urgent need for genetic diagnosis

What is Rapid PGxome Diagnostic?

PGxome is PreventionGenetics' whole exome sequencing (WES) test. The PGxome assesses almost all genes from the human genome including coding regions and adjacent introns. This test is an appropriate choice for health care providers who are looking for an urgent genetic diagnosis. This is important as more than 50% of patients with genetic diseases are not given a specific diagnosis even after repeat clinical examinations and tests (Shashi et al. 2014). The standard clinical practice often involves examinations for specific phenotypes, imaging, biochemical testing for inborn errors of metabolism, genomic tests such as karyotyping or microarrays, and single gene or panel tests (Iglesias et al. 2014). However, patients remain without a genetic diagnosis, and patients and health care providers are caught in a long term diagnosis search, known as a diagnostic odyssey. This can lead to failures in identifying potential treatments, and unknown recurrence and prognosis risks (Yang et al. 2013).

Reported diagnostic rates from commercial and academic laboratories have found that WES assays have a ~20-40% positive diagnostic rate, with higher rates being reported from trio analysis (i.e. proband and parents) compared to singleton analysis (Atwal et al. 2014; Iglesias et al. 2014; Farwell et al. 2015). Notably, ~5-7% of individuals who have WES have had dual diagnoses (i.e. two non-overlapping clinical presentations) (Yang et al. 2014; Farwell et al. 2015; Posey et al. 2016). The inclusion of copy number variant (CNV) calling should increase diagnostic rates. One study reported that 30% of genetics diagnoses have only been recently resolved due to new literature reports, highlighting the fast pace of gene-disease discovery and the need of genetic testing laboratories to be current of the medical literature (Yang et al. 2014). The use of a whole exome sequencing test may aid in altering clinical management, predict recurrence and prognosis risks, and reduce costs of additional testing, and may offer advantages over traditional molecular tests in certain patients (Valencia et al. 2015).

Rapid PGxome - Diagnostic is ideal for:

  • Seriously ill patients with an urgent need for genetic diagnosis

TURN AROUND TIME (TAT)

Rapid PGxome has an expedited TAT of 14 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 Rapid PGxome 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, exome sequencing is performed only on the patient’s specimen, and depending on variants identified specifically related to the patient’s clinical presentation, parental specimens are then used for targeted testing to determine the phase of variants or to determine if a variant occurs de novo. For the highest diagnostic rate, Family - Trio testing is recommended.

Specimen Requirements and Shipping Details

Specimen TypeSpecimen Requirements
Blood

Draw blood in EDTA (purple-top) or ACD (yellow-top) tubes.

  • Infants <2 years old: 1mL-2mL blood in EDTA or ACD tubes
  • Adults and children >2yrs: 3mL-5mL blood in EDTA or ACD tubes
DNASend in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 ng/µl.
Tissue (Fresh frozen)Fresh frozen tissue accepted. Please contact us for details. Fixed, paraffin embedded samples are not accepted.
Cultured CellsShip at least two T25 flasks of confluent cells.

Specimen quality is of utmost importance for rapid testing. At this time, saliva, buccal, and direct amniocytes/CVS are not accepted.

Storage and shipping

T25 flasks need to be shipped in appropriate complete culture medium filled to the brim. Ship T25 flasks at room temperature in an insulated container once 90% confluency is reached and by overnight delivery.

TEST METHODS

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. 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. Common benign, likely benign, and low quality variants are filtered from analysis.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All reported CNVs are confirmed using another technology such as aCGH, MLPA, or PCR.

REPORTING

Only primary findings will be reported for Rapid PGxome. Reports will consist of two sections:

  1. Variants in genes known to be associated with phenotype
  2. Variants in genes possibly associated with phenotype

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

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). Only relevant Pathogenic, Likely Pathogenic, and Uncertain variants are reported. Likely benign and benign variants are not included in the reports. 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.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants relevant to the patient's health will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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

Copy Number Variant Analysis: The PGxome test detects most deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

The sensitivity of this test is dependent on DNA quality.

CONTACTS

Genetic Counselors: GC Team - support@preventiongenetics.com

Geneticist: Diane Allingham-Hawkins, PhD, FCCMG, FACMG - diane.allingham-hawkins@preventiongenetics.com

REFERENCES

Atwal P.S. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 717-9. PubMed ID: 24525916

Caudle et al. 2016. Genetics in Medicine. PubMed ID: 27441996

Farwell K.D. et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 578-86. PubMed ID: 25356970

Iglesias A. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 922-31. PubMed ID: 24901346

Kalia S.S. et al. 2016. Genetics in Medicine: Official Journal of the American College of Medical Genetics. Advance online publication. doi:10.1038/gim.2016.190. PubMed ID: 27854360

Posey et al. 2016. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 18: 678-85. PubMed ID: 26633545

Richards S et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 405-24. PubMed ID: 25741868

Shashi V. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 176-82. PubMed ID: 23928913

Valencia C.A. et al. 2015. Frontiers in Pediatrics. 3: 67. PubMed ID: 26284228

Yang Y. et al. 2013. The New England Journal of Medicine. 369: 1502-11. PubMed ID: 24088041

Yang Y. et al. 2014. JAMA. 312: 1870-9. PubMed ID: 25326635