Test Methods

Test Procedure

For PGxome® we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using 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 >100x. Data analysis and interpretation is performed by the internally developed Infinity pipeline. Variant calls are made by the GATK haplotype caller (through Sentieon) and annotated using in house software and Jannovar.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

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 CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

NextGen Sequencing: As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Copy Number Variant Analysis: The PGxome test detects most larger 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.

Analytical Limitations

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.

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 will not be detected.

We sequence coding exons for all available transcripts plus 10 bp of flanking non-coding DNA for each exon. We also sequence other regions within or near genes in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere.  Unless specifically indicated, test reports contain no information about other portions of genes.

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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

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

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 sample 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.

Exome-Wide Copy Number Variant (CNV) Analysis is available as an add-on order with the request of any PGxome-based or custom panel. To confirm if this is an option for your order, visit the panel-specific description on our website. Unavailable for PG-Select panels, Sanger sequencing, and other test methods.

What is Exome-Wide CNV Analysis?

PreventionGenetics’ Exome-Wide CNV Analysis utilizes exome sequencing data to identify chromosomal imbalances similar to those detected by chromosomal microarray (CMA). Our algorithm has been extensively validated to ensure appropriate identification of aneuploidy, triploidy, unbalanced rearrangements (e.g., unbalanced translocations), and known microdeletion and microduplication syndromes. This method will also identify unique CNV events. This add on option allows for cost-effective identification and reporting of potentially important, large CNVs across the full exome in conjunction with any sequencing variants and/or smaller CNVs identified within the panel ordered. Exome-Wide CNV results will be included in the panel test report.

Test Procedure

Copy Number Variants (CNVs) are 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. For Exome-Wide CNV Analysis, we will generally only report deletions ≥ 250 kb in size and duplications ≥ 500 kb in size.

A written summary and interpretation of the CNVs are provided using standard aCGH nomenclature and in line with the ACMG guidelines for microarray interpretation. Common CNVs and rare CNVs with unknown clinical significance may be detected with this technology. CNVs in regions of the genome with no clinically relevant genes or in regions reported as common polymorphism in the general population will not be reported. In addition, CNVs confined to deep intronic sequences with no documented evidence of clinical significance will not be reported.

For CNVs with unknown clinical significance, a number of considerations are taken into account, including the size, the gene content, whether the CNV is a deletion or a duplication, and the inheritance pattern. On some occasions, interpretation of whether a CNV is pathogenic depends on whether the CNV is inherited from one of the parents or de novo, and hence parental analysis is sometimes needed for optimal interpretation.

For CNV results indicative of a structural chromosomal abnormality, reflexive chromosome studies may be warranted for the patients and/or parents to guide recurrence risk estimation.

Analytical Limitations

This test targets deletions larger than 250 kb and duplications larger than 500 kb. In rare cases, sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size, and inadequate coverage may impact our ability to identify and/or interpret a CNV.

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.

The majority of PreventionGenetics Next-Gen sequencing panels are orderable using PGnome (genome) sequencing. This option allows for improved, more uniform coverage, better structural variant (SV) calling, and assessment of deep intronic regions (where applicable) with the same turnaround time at a slightly increased test cost. Visit the test-specific description to learn if PGnome sequencing is an available option.

At this time, only whole blood or DNA from whole blood are accepted for PGnome sequencing panels.

Test Procedure

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.

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. When warranted by sequence results (for example a single pathogenic variant in a recessive gene), we examine all rare variants within selected gene 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.

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.

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, duplications, insertions, and inversions. 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.

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.

Analytical Limitations

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. The three SV calling algorithms that we employ (Lumpy, CNVnator, and Manta) 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. Our 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, we are not reporting translocations. Our ability to detect SVs due to somatic mosaicism is limited.

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://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).

Interpretation of the test results is limited by the information that is currently available. Better interpretation will be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

Genome-Wide Structural Variant (SV) Analysis is available as an add-on order with the request of any PGnome-based panel. To confirm if this is an option for your order, visit the panel-specific description on our website. Unavailable for PG-Select panels, Sanger sequencing, and other test methods.

What is Genome-Wide SV Analysis?

PreventionGenetics’ Genome-Wide Structural Variant (SV) Analysis utilizes genome sequencing data to identify chromosomal imbalances similar to those detected by chromosomal microarray (CMA). Our algorithm has been extensively validated to ensure appropriate identification of aneuploidy, triploidy, unbalanced rearrangements (e.g., unbalanced translocations), and known microdeletion and microduplication syndromes. This method will also identify unique SV events. This add-on option allows for cost-effective identification and reporting of potentially important, large SVs across the full genome in conjunction with any sequencing variants and/or smaller SVs identified within the panel ordered. Genome-wide SV results will be included in the panel test report.

Test Procedure

Structural variants (SVs) are detected from NGS data. The three SV calling algorithms that we employ (Lumpy, CNVnator, and Manta) 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. Our 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, we are not reporting translocations. Our ability to detect SVs due to somatic mosaicism is limited.

All reported SVs are confirmed using another technology such as aCGH, MLPA, or PCR. For Genome-Wide SV Analysis, we will generally only report deletions ≥ 250 kb in size and duplications ≥ 500 kb in size

A written summary and interpretation of the SVs are provided using standard aCGH nomenclature and in line with the ACMG guidelines for microarray interpretation. Common SVs and rare SVs with unknown clinical significance may be detected with this technology. SVs in regions of the genome with no clinically relevant genes or in regions reported as common polymorphism in the general population will not be reported. In addition, SVs confined to deep intronic sequences with no documented evidence of clinical significance will not be reported.

For SVs with unknown clinical significance, a number of considerations are taken into account, including the size, the gene content, whether the SV is a deletion or a duplication, and the inheritance pattern. On some occasions, interpretation of whether a SV is pathogenic depends on whether the SV is inherited from one of the parents or de novo, and hence parental analysis is sometimes needed for optimal interpretation.

For SV results indicative of a structural chromosomal abnormality, reflexive chromosome studies may be warranted for the patients and/or parents to guide recurrence risk estimation.

Analytical Limitations

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, duplications, insertions, and inversions. This test targets deletions larger than 250 kb and duplications larger than 500 kb.

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.

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.

The sensitivity of this test is dependent on DNA quality.

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Deletion and Duplication Testing via NGS

Analytical Validity

NextGen Sequencing

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Analytical Limitations

NextGen Sequencing

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.

When 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 amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

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 PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes 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.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

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 sample arrives at PreventionGenetics.

Test Procedure

As required, genomic DNA is extracted from the patient specimen prior to amplification and sequencing. For PGmito, we use long-range polymerase chain reaction (lr-PCR) followed by massively parallel, high-throughput sequencing (next generation sequencing) in order to accurately detect heteroplasmic mtDNA variants. To mitigate allele dropout, two separate PCR primer sets are used to amplify the entire mtDNA genome, resulting in two separate (redundant) fragments for analysis. For NGS, 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: 100% of targeted bases are covered at a minimum of 3000x. Variant calls are made by the MutServe pipeline. Next generation sequencing and subsequent pipeline analysis allows for accurate detection of low-level heteroplasmic (down to ~4%) variants in patient samples.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Analytical Validity

Twenty-three well-characterized controls were used to validate this assay. When both fragments were analyzed for each sample, we accurately detected all reported variants down to 4% heteroplasmy, with the great majority of calls (91%) within 1% of the heteroplasmic load reported by the source (if known). Variants above 20% were Sanger confirmed; variants <20% heteroplasmy were not analyzed in Sanger given the limit of detection for that methodology. This analysis involved ~1,000 total sequence variants (differences from the reference sequences), with the vast majority (>99.8%) being single nucleotide substitutions.

Additionally, mixing experiments were performed utilizing samples from two individuals with multiple different mitochondrial homoplasmic variants. These two samples were combined in a series of different ratios to artificially mimic heteroplasmy (down to 1%). When both fragments were analyzed, all variants were accurately detected for each sample down to 4% heteroplasmy.

Analytical Limitations

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.

Although two separate primer sets are utilized with this test to help mitigate allele dropout, a patient may occasionally carry a proportion of mtDNA genomes which do not amplify or capture, due for example to a large deletion or insertion, or single nucleotide polymorphisms that lie underneath our primer set(s) and prevent primer binding.

We sequence essentially the entire mtDNA genome.  However, largely due to runs of tandem repeats in non-coding regions, a few small regions are excluded from analysis (m.280-m.320, m.490-m.575, m.3106-m.3107, m.12418, and m.16150-m.16200). This test contains no information regarding variants in these regions.

The ability to accurately detect extremely low-level heteroplasmy (<4%) is limited, and therefore we will not report out on potential heteroplasmic variants that are <4% heteroplasmy.

Test reports contain no information about the DNA sequence in other cell types, yet heteroplasmic load may vary significantly between different tissue types in the same individual. When feasible, affected tissue types should be tested to provide the most relevant sequence results.

Sanger confirmation is attempted on all reportable sequence variants regardless of heteroplasmic load. However, due to technical limitations, variants <20% heteroplasmy may fall below the limit of detection for Sanger sequencing. If we are unable to confirm a variant in Sanger, the variant will be listed on reports with a limitation regarding the lack of confirmation with a secondary methodology. Testing an alternate tissue type may be necessary to clarify the finding. 

We cannot be certain that the reference sequences are correct.  The revised Cambridge Reference Sequence (rCRS) is used as our reference.

Detection of gross deletions, duplications, or large rearrangements is not currently available for this test.

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 sample 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.

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://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). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.Benign variants are not listed in the reports, but are available upon request. 

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

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.

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

Only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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.

We cannot be certain that the reference sequence(s) are correct.  Exons, for example, may be misidentified.  In cases where the genomic and mRNA sequences disagree, we use the genomic sequence as our reference.

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

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

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

In the case of duplications, aCGH will not determine the chromosomal location of the duplicated DNA. Most duplications will be tandem, but in some cases the duplicated DNA will be inserted at a different locus. This method will also not determine the orientation of the duplicated segment (direct or inverted).

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay is dependent upon the quality of the input DNA. In particular, highly degraded DNA will yield poor results.

Test Procedure

CMA starts with the extraction of genomic DNA from a patient specimen (e.g., whole blood) using a QIAamp DNA Blood Midi kit (Qiagen). Equal amounts of genomic DNA (~1.0 microgram) from a patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. Labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto a microarray slide and hybridized for at least 22-24 hours at 65°C. Arrays are then washed and scanned immediately at 2.5 µM resolution.

Analytical Validity

CMA copy number data is analyzed following feature extraction of the scanned microarray, using Agilent CytoGenomics Software and in-house tools. The resolution for CNV detection depends on the region of the genome, its uniqueness and complexity. On average, across the targeted regions, this array can detect CNVs of minimal size between 500 bp to 30 kb.

Analytical Limitations

CMA can only detect only gross genomic copy number imbalances in the nuclear genome.

CMA cannot detect:

• Balanced chromosomal rearrangements such as inversions, balanced insertions, and reciprocal translocations
• Genomic copy number changes in the regions of the genome not represented on the microarray (including regions with repeat sequences such as segmental duplications, repeat sequences in the short arms of acrocentric chromosomes, and heterochromatic regions)
• Low levels of mosaicism for regions
• Single nucleotide variants and small insertions or deletions
• Imbalances in the mitochondrial genome
• Imbalances when mosaicism for reciprocal CNVs exist. For example, when mosaicism for 46,XX, 45,X and 47XXX exist in approximate equal proportions, CMA will fail to detect the presence of the clinically significant 45,X line
• Failure to detect an alteration at a specific locus does not rule out the diagnosis of a genetic disorder associated with that locus. Other abnormalities may be present that are undetectable by the microarray design

Test Procedure

CMA starts with the extraction of genomic DNA from a patient specimen (e.g., whole blood) using a QIAamp DNA Blood Midi kit (Qiagen). Equal amounts of genomic DNA (~1.0 microgram) from a patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. Labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto a microarray slide and hybridized for at least 22-24 hours at 65°C. Arrays are then washed and scanned immediately at 2.5 µM resolution.

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

CMA copy number data is analyzed following feature extraction of the scanned microarray, using Agilent CytoGenomics Software and in-house tools. The resolution for CNV detection depends on the region of the genome, its uniqueness and complexity. On average, across the targeted regions, this array can detect CNVs of minimal size between 500 bp to 30 kb.

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

CMA can only detect only gross genomic copy number imbalances in the nuclear genome.

CMA cannot detect:

• Balanced chromosomal rearrangements such as inversions, balanced insertions, and reciprocal translocations
• Genomic copy number changes in the regions of the genome not represented on the microarray (including regions with repeat sequences such as segmental duplications, repeat sequences in the short arms of acrocentric chromosomes, and heterochromatic regions)
• Low levels of mosaicism for regions
• Single nucleotide variants and small insertions or deletions
• Imbalances in the mitochondrial genome
• Imbalances when mosaicism for reciprocal CNVs exist. For example, when mosaicism for 46,XX, 45,X and 47XXX exist in approximate equal proportions, CMA will fail to detect the presence of the clinically significant 45,X line
• Failure to detect an alteration at a specific locus does not rule out the diagnosis of a genetic disorder associated with that locus. Other abnormalities may be present that are undetectable by the microarray design

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Test Procedure

See methodology section in the supplemental information included in test reports.

Test Procedure

As required, genomic DNA (gDNA) is extracted from the patient specimen. gDNA extracted from blood samples/submitted DNA from the patient is denatured and hybridized to MLPA probes specific to exonic or intronic regions of a particular gene(s). Each probe consists of two adjacent oligonucleotides that once hybridized to patient/reference DNA are ligated into a single DNA fragment.  Fluorescently labeled PCR is then used to amplify each ligated probe using a common PCR primer set. The amplicon is then sized using a capillary electrophoresis instrument. The relative peak height of each amplified probe from the patient’s sample is compared to four internal negative controls to determine relative copy number.  For each patient sample the data for only the gene(s) of interest is analyzed and reported.

Analytical Validity

MLPA enables the detection of relatively small deletion and insertion variants within a single exon of a given gene or within an entire gene.

Analytical Limitations

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.

Only the indicated gene or genes are analyzed. Test reports contain no information about other regions of the genome, including genes that are not requested, and genes that are not targeted. This test does not provide any information about deletions or duplications within repetitive elements.

Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.

We cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we cannot determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.

For a single probe deletion or duplication we will compare MLPA results to sequencing results to ensure that no single nucleotide polymorphisms are underlying the specific probe, which may affect probe hybridization.

Any partial exonic deletions and duplications outside the probe binding sequence cannot be detected.

Impurities in the test and reference DNA samples can increase the chance of false positive or negative results. Where possible similar DNA extraction methods between test and reference samples are ideal for relative copy number analysis.

Our ability to detect minor copy number change, due for example to somatic mosaicism may be limited. 

Unless otherwise indicated, MLPA results are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about copy number changes in other tissues. 

We cannot be certain that the reference sequence(s) are correct. Exons, for example, may be misidentified.

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 sample arrives at PreventionGenetics. 

Normal findings within a targeted gene do not rule out the clinical diagnosis of a genetic disease.

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

Test Procedure

Multiplex Ligation-dependent Probe Amplification (MLPA) is a semi-quantitative technique that is used to determine the relative copy number of up to 60 DNA sequences in a single multiplex PCR-based reaction. It is based on amplification of up to 60 probes, each of which detects a specific complementary DNA sequence of approximately 60 bp in length (often exons in genes of interest). Briefly, each MLPA probe is made up of two half-probes that hybridize immediately adjacent to each other at the target DNA. These adjacent probes are then ligated into one single probe before being amplified in a PCR reaction. Multiplexing is achieved by different probes varying in sizes ranging from 150-500 bp, that are all amplified using a common PCR primer pair. One of the PCR primers is fluorescently labelled enabling separation and detection of the amplification products in a capillary electrophoresis instrument. The peaks heights of the amplification products of the target DNA sequence is then compared to the peak heights in various reference DNA samples. A deletion or a duplication is inferred from the relative decrease or increase in peak height respectively.

A modified MLPA technique termed Methylation-specific MLPA (MS-MLPA) is used to detect both the copy number and methylation status of up to 50 DNA sequences in a single multiplex PCR-based reaction. The basic principle of MS-MLPA is very similar to MLPA except that the target DNA sequences recognized by the MS-MLPA probes contain restriction sites for enzymes such a HhaI or HpaII that are sensitive to cytosine methylation of one CpG site in their recognition sequence. When target DNA is digested with these enzymes a probe amplification product will only be obtained if the CpG site is methylated. The level of methylation is determined by the ratio of the relative peak area for each target probe from digested vs undigested DNA sample.

Analytical Validity

MS-MLPA is a robust method that is widely used for the clinical diagnosis of several genetic imprinting disorders like Prader-Willi syndrome /Angelman syndrome, Beckwith-Widemann syndrome, Russell-Silver syndrome, Lynch syndrome and Albright hereditary osteodystrophy. MS-MLPA has several advantages over other assays such as MS-PCR based on bisulphite sequencing, southern blotting, and methylation analysis including PCR following restriction digestion with methylation sensitive enzyme. MS-MLPA investigates methylation status at multiple loci, thereby reducing the risk for false positive or false negative results due single nucleotide polymorphisms (SNPs) at the probe binding sequence.

Analytical Limitations

Both MLPA and MS-MLPA will not detect point mutations in sequences recognized by the probes. In addition it will not detect inversions, balanced translocations or copy number changes that lie outside the sequence detected by the MLPA probes.

MLPA probes are sensitive to changes within the sequence detected by the probe. A single nucleotide change (such as SNPs or pathogenic mutations) very close to the probe ligation site can prevent ligation of the two oligonucleotide probes. In addition, sequence changes further from the ligation site can affect probe binding and hence decrease probe signal mimicking a deletion.

MLPA is sensitive to DNA characteristics such as impurities, method used for DNA isolation, salt concentrations in solution, and degree of DNA degradation.  The effect of these characteristics can be minimized by using the same DNA extraction methods for all samples analyzed by this method and by matching the test and control DNA from the same source.

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

gDNA extracted from blood samples/submitted DNA from the patient is denatured and hybridized to MLPA probes specific to exonic or intronic regions of a particular gene(s). Each probe consists of two adjacent sequences that once hybridized to patient/reference DNA are ligated into a single probe.  Fluorescently labeled PCR is then used to amplify each ligated probe using a common PCR primer set. The amplicon is then visualized during fragment separation using a capillary electrophoresis instrument. The relative peak height of each amplified probe from the patient’s sample is compared to four internal negative control results to determine relative copy number.  For each patient sample the data for only the gene(s) of interest is analyzed and reported.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error. Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

MLPA enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele. Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences. In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions. In nearly all 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 for example 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 PCR and cycle sequencing. Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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.

Only the indicated gene or genes were analyzed. Test reports contain no information about other regions of the genome, including genes that are not requested, and genes that are not targeted. This test does not provide any information about deletions or duplications within repetitive elements.

Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.

We cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we cannot determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.

For a single probe deletion or duplication we will compare MLPA results to sequencing results to ensure that no single nucleotide polymorphisms are underlying the specific probe, which may affect probe hybridization.

Any partial exonic deletions and duplications outside the probe binding sequence cannot be detected.

Impurities in the test and reference DNA samples can increase the chance of false positive or negative results. Where possible similar DNA extraction methods between test and reference samples are ideal for relative copy number analysis.

Our ability to detect minor copy number change, due for example to somatic mosaicism may be limited. 

Unless otherwise indicated, MLPA results are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about copy number changes in other tissues. 

We cannot be certain that the reference sequence(s) are correct. Exons, for example, may be misidentified.

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 sample arrives at PreventionGenetics. 

Normal findings within a targeted gene do not rule out the clinical diagnosis of a genetic disease.

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

Test Procedure

As required, DNA is extracted from the patient specimen.

Repeat-primed PCR

The size of the CGG repeat in the FMR1 gene is determined using an AmplideX  FMR1 PCR kit from Asuragen (Filipovic-Sadic et al. 2010). This kit uses repeat-primed PCR to detect both normal and expanded CGG repeat alleles in the FMR1 gene. The PCR is performed with the following three types of primers:

- Two FMR1 specific primers that flank the CGG repeat region. The reverse primer is fluorescently labeled.

- A forward PCR primer that is complimentary to the CGG repeat. This primer can anneal at multiple sites along the repeat, resulting in a mixture of PCR products of increasing size. These PCR products are called CGG repeat primed peaks.

The two gene specific primers amplify full-length PCR products from each allele. The addition of the third repeat primer allows for detection of CGG repeat primed peaks, spanning from the smallest allele to the largest allele. The presence of repeat primed peaks allow for the detection of a very large CGG expansion, even if a gene specific peak is not detected to PCR dropout.

All PCR products are run on an ABI3730xl sequencer.

Methylation specific PCR

Methylation status of expanded alleles is assessed using an AmplideX  FMR1 mPCR kit from Asuragen (Filipovic-Sadic et al. 2010).  This kit pairs methylation sensitive DNA digestion with  FMR1 specific PCR amplification of the CGG repeat region to determine the methylation status of full mutation alleles.

Analytical Validity

A total of twenty-two samples were used for test validation. These include:

- Two DNA samples from Asuragen
- Ten DNA samples from Coriell Cell Respositories:
- 13 DNA samples previously tested at other laboratories

Control DNA samples from Coriell Cell Respositories:

- NA07538, NA07862, NA06896, NA07543, NA06891, NA06852 , NA06968, NA20234, NA20239, NA18310

Analytical Limitations

The repeat-primed PCR assay is not designed to determine the exact number of repeats in alleles containing over 200 repeats (full mutations). This test has been validated to detect up to 10% mosaicism. Only the methylation status of Full Mutations (>200 CGG repeats) is determined.

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.

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 sample arrives at PreventionGenetics.

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

Test Procedure

As required, DNA is extracted from the patient specimen.

Repeat-primed PCR

The repeat-primed PCR assay is used to determine the presence or absence of a nucleotide repeat expansion, as previously described (Warner et al. 1996; Renton et al. 2011; DeJesus-Hernandez et al. 2011; Jama et al. 2013). PCR is performed with a fluorescently labeled unique forward primer specific to the gene of interest. The reverse primer is specific to the repeat region allowing for annealing at multiple locations within the repeat region. Due to the multiple annealing sites, amplicons will vary in size according to the number of nucleotides in the repeat. A tail anchored primer may also be used in conjunction with the reverse primer to minimize progressive shortening of amplicons.

PCR products are then analyzed on an ABI3730xl sequencer.

Fluorescent Fragment-length Assay

The purpose of this assay is to confirm results obtained from the repeat-primed PCR, using flanking primers specific to the gene repeat of interest. The PCR is carried out using a fluorescently labeled forward primer and a reverse primer which flank the repeat of interest as described (DeJesus-Hernandez et al. 2011; Jama et al. 2013). PCR products are then analyzed on an ABI3730xl sequencer. Two peaks will be observed in patients with two different sized alleles. In patients with a very large expansion, it is possible that only one allele will be observed. The repeat-primed PCR will determine if the patient is a true homozygote or if they have a large expansion.

Analytical Validity

A variety of positive and negative control DNA samples from affected and unaffected individuals were used to validate this test. Controls spanned the full range from the smallest normal alleles to near the largest expansion that has been reported.

Analytical Limitations

The repeat-primed PCR assay may or may not be designed to determine the number of repeats in alleles carrying the pathogenic expansion. Please see individual gene test descriptions for details.

Test Procedure

As required, DNA is extracted from the patient specimen.

Sanger Sequencing

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants

(2) Likely Pathogenic Variants

(3) Variants of Uncertain Significance

(4) Likely Benign Variants

(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Fluorescent Fragment-length Assay

The purpose of this assay is to confirm results obtained from bidirectional Sanger sequencing, using flanking primers specific to the gene repeat of interest. The PCR is carried out using a fluorescently labeled forward primer and a reverse primer which flank the repeat of interest as described (Brais et al. 1998. PubMed ID: 9462747; Richard et al. 2017. PubMed ID: 28011929). PCR products are then analyzed on an ABI3730xl sequencer. Two peaks will be observed in patients with two different sized alleles, while a single peak will be observed in patients with two alleles of the same size. Barring any technical issues, the Sanger sequence results will help confirm that the patient is a true homozygote (see Analytical Limitations, below).

Analytical Validity

Regarding Sanger sequencing, as of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable.

Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Regarding the fluorescent fragment-length assay, a variety of positive and negative control DNA samples from affected and unaffected individuals were used to validate this test. Controls spanned the full range from the normal (GCN)10/Ala10 allele to a (GCN)13/Ala13 allele.

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not bedetected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n &gt;8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Test Procedure

As required, DNA was extracted from the patient specimen.  Long-range Polymerase Chain Reaction (lr-PCR) is used to amplify the mitochondrial genome.  To help mitigate allele dropout, two separate PCR primer sets are used to amplify the entire mitochondrial genome, resulting in two separate (redundant) fragments for analysis. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.1 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, cycle sequencing is performed separately in both the forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.

Human Genome Variation Society (HGVS) recommendations are used to name sequence variants (Human Genome Variation Society). 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). mtDNA sequence variants are interpreted utilizing a combination of resources, including recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (Richards et al. 2015. PubMed ID: 25741868), which was primarily developed for interpretation of variants in nuclear genes; as well as recommendations from the MSeqDR consortium (McCormick et al. 2020. PubMed ID: 32906214) and Baylor College of Medicine (Wong et al. 2020. PubMed ID: 31965079), which were developed specifically for mtDNA variant interpretation.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

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 improves.

When sequencing did not reveal any differences from the reference sequence, we cannot be certain that we were able to PCR amplify all patient alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or insertion. In these cases, the report contains no information about the additional allele. Our Sanger sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

Only the indicated targeted variant(s) are analyzed. Test reports contain no information about other portions of the mitochondrial genome.

Our ability to detect low-level heteroplasmic variants (<20%) using this methodology is limited.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific cell type (usually leukocytes). Test reports contain no information about gene sequences in other cell types.

We cannot be certain that the reference sequence(s) are correct. The revised Cambridge reference sequence (rCRS) is currently used as our reference for the mitochondrial genome.

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

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