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High-quality genetic testing doesn't need to be expensive

Genetic test results you can trust

To demonstrate that Invitae's next-generation sequencing (NGS) analysis provides the high-quality results you are accustomed to, Invitae has validated our analytic results and clinical interpretations through a number of studies:

Invitae hereditary cancer validation study

A systematic comparison of traditional and multi-gene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients


A study comparing Invitae’s panel test to traditional BRCA1 and BRCA2 tests in more than 1000 patients was undertaken in collaboration with the Stanford University School of Medicine and Massachusetts General Hospital. The study demonstrated 100% analytic sensitivity and specificity for Invitae’s panel compared to traditional genetic test results for both sequence alterations and deletions/duplications. Variant classifications were also highly (99.8%) concordant.


Multi-gene panels for hereditary breast and ovarian cancer risk assessment are gaining acceptance, not only as additions to but also as replacements for traditional BRCA1/2 testing. To help determine which tests are appropriate for any given patient, it is important to understand the analytic and clinical performance of these tests by comparison with traditional testing.


A total of 1105 individuals were tested using an Invitae 29-gene hereditary cancer panel. Sequence alterations and copy number deletions/duplications were determined by next-generation sequencing (NGS) using Invitae’s custom biochemical and bioinformatics methodologies. For these 1105 individuals, high-quality reference and confirmatory data were available for direct comparison. Variants were classified using a framework (Sherloc) based on the American College of Medical Genetics and Genomics 2015 guidelines using only publicly available and not proprietary data resources. Classifications were compared for 975 individuals for whom traditional BRCA1/2 test results from Myriad Genetics were available.

Table 1: Analytic concordance

Table 1: Analytic concordance

Figure 1: Types of pathogenic variants observed

Figure 1: Types of pathogenic variants observed

Table 2: Interpretation concordance for BRCA1/2

Table 2: Interpretation concordance for BRCA1/2



  • 100% analytic sensitivity and specificity was observed across all 750 comparable variant calls in the 1105 individuals.
  • These 750 variants included 48 technically challenging examples of sequence and/or copy number variation that together represented a significant fraction (13.4%) of the pathogenic variants in the prospective cases.
  • Considering variant classifications for BRCA1/2, 99.8% report concordance was observed.
  • The rates of variants of uncertain significance for BRCA1/2 testing were comparable, albeit slightly higher, in the Invitae test versus the traditional tests (4.1% vs. 3.2%).
  • Consistent with other studies of comparable populations, 4.5% of the BRCA1/2-negative patients had a mutation uncovered in another cancer risk gene.


Invitae’s NGS panel test can provide analytic and clinical results highly comparable to those of traditional BRCA1/2 testing. For both sequence and deletion/duplication variants across many genes, 100% sensitivity and specificity was observed, as well as high interpretation concordance (99.8%). Panel tests can also uncover potentially actionable findings that may be otherwise missed. A detailed study of the clinical actionability of non-BRCA1/2 variants observed in these and other patients is reported separately.


This study is published in the Journal of Molecular Diagnostics, the official journal of the Association for Molecular Pathology.

Stephen E Lincoln, Yuya Kobayashi, Michael J Anderson, Shan Yang, Andrea J Desmond, Meredith A Mills, Geoffrey B Nilsen, Kevin B Jacobs, Federico A Monzon, Allison W Kurian, James M Ford, Leif W Ellisen, A systematic comparison of traditional and multi-gene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients. J Mol Diagn. 2015.

Download the Invitae hereditary cancer analytic validation one-page PDF of this information.

Invitae confirmation for clinical genetic testing

Current clinical approach

For decades, Sanger sequencing has been accepted by clinicians as a gold-standard in genetic testing. However, its low throughput is not ideal for assessing the growing number of genes in a modern clinical gene panel. When clinical testing began to employ next-generation sequencing (NGS), a two-step approach was adopted whereby many genes were assayed by NGS sequencing and reportable variants were verified by Sanger sequencing. This continues to be the standard as set out by the ACMG guidelines: “... it is recommended that all disease-focused and/or diagnostic testing include confirmation of the final result using a companion technology.”1

It has since been reported that Sanger sequencing may introduce more errors than it actually prevents, and may be unnecessary for high quality variants.2,3,4 Here we examine the results of this two-step confirmatory approach as it has been implemented in our laboratory.

Quality in confirmation

For reasons both of efficiency and accuracy, Invitae this year validated Pacific Biosciences (PacBio) sequencing technology for clinical use. It is an alternate confirmatory sequencing method that, in our hands, provides an accurate and higher throughput method that is orthogonal to NGS sequencing. PacBio provides a more quantitative measurement of variants and additional QC metrics compared to Sanger sequencing. We continue to use both PacBio and Sanger to confirm variants.

Confident NGS variant calls are highly specific

Using this hybrid method of NGS followed by either Sanger or PacBio to deliver tens of thousands of clinical reports, Invitae has built a large dataset of variants detected by all three technologies. This has allowed us to complete a direct comparison of NGS with the two orthogonal technologies (see table below). Standard orthogonal confirmation assays rely on PCR-based targeting of the variant loci, which is susceptible to allele dropout and amplification failure. To guard against a false negative result due to these types of failures, we run multiple overlapping assays to redundantly target each variant.

In a cohort of approximately 70,000 individuals who have undergone genetic testing at Invitae, we identified nearly 6,800 high quality variant calls that because of our reporting policies required orthogonal confirmation. Our data show that among 6,788 variants (57% SNVs and 43% indels) detected with high confidence according to our QC parameters, 6,752 (99.5%) were confirmed by Sanger or PacBio sequencing. We have never observed a discordant result during the orthogonal confirmation process of high confidence variant calls. For the remaining 36 (0.5%) high confidence variants detected by NGS, the orthogonal assay generated no useable data after several attempts and the variant was reported with a corresponding limitation regarding the failure to confirm according to ACMG guidelines. These events tend to be in regions known to be difficult for these assays, such as areas of high GC content or within repetitive sequences.

Table: Next-generation sequencing and orthogonal methods show perfect concordance for high quality variant calls

Table: Perfect concordance for high quality variant calls

False positive rate and sensitivity

In order to maintain high sensitivity, a wide net must be cast when initially calling variants. Our validated, custom computational pipeline identifies variants and assesses their quality based on a large number of sequencing attributes (including strand bias, read depth, Phred-scaled p-value, and many others). These quality attributes are used to separate variants into those that are high-quality and others that may be false positives and are “flagged” for further evaluation and mandatory confirmatory testing.

In the same testing period in which the 6,788 high quality variants were found, 508 low-quality, flagged variants were also detected and, among these, 411 (81%) confirmed as true positives and 97 (19%) were found to be false positives by an orthogonal method. Thus, out of 7,296 total variants, 1.3% (97 of 7,296) were NGS false positives that were correctly identified during confirmatory testing.


Our data show that confirmatory sequencing has not improved the accuracy of the final report for high quality NGS variant calls. It does however continue to be important when the quality metrics for the variant do not exceed empirically determined thresholds set in our lab.

N.B.: Although it is outside the scope of this white paper, note that Invitae confirms any reported CNV event and has performed more than 1,000 such confirmations.

Next-generation sequencing, as implemented at Invitae, is a high-quality, clinical grade technology. Our team understands that the stakes for clinical genetic testing are high. Results can lead to irreversible action and emotional distress for both the patient and their family. We are committed to maintaining the highest quality, while continually improving our processes in a responsible and data driven manner.


1. Rehm et al. ACMG clinical laboratory standards for next-generation sequencing. Genet. Med. 2013
2. Beck et al. Systematic evaluation of Sanger validation of next-generation sequencing variants. Clin. Chem. 2016
3. Baudhuin et al. Confirming variants in next-generation sequencing panel testing by Sanger sequencing. JMD 2015
4. Mu et al. Sanger confirmation is required to achieve optimal sensitivity and specificity in next-generation sequencing panel testing. JMD 2016

Download the Invitae confirmation for clinical genetic testing one-page PDF of this white paper.

Sequencing and deletion/duplication analysis of exons 12-15 of PMS2 using next-generation sequencing (NGS)


Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is characterized by familial predisposition to cancers of the colon, endometrium, ovary, stomach, and urinary tract.1 Most cases of Lynch syndrome are caused by variants in MLH1, MSH2, and MSH6, but 4–11 percent of cases are caused by variants in PMS2.2-4

Testing for inherited variants in PMS2 is hampered by the presence of a pseudogene, PMS2CL, which has nearly identical homology to PMS2 in the final four exons of the gene (exons 12–15). Thus, sequence reads derived from hybridization capture in next-generation sequencing (NGS) methods cannot be unambiguously aligned to PMS2 or PMS2CL. Gene conversion between exons 12 and 15 of PMS2 and PMS2CL further complicates this issue.5

Why develop an NGS method?

Invitae is committed to making high-quality genetic testing affordable and accessible. Most laboratories perform multiplex ligation-dependent probe amplification (MLPA) to identify deletion/duplication variants, and use long-range PCR (LR-PCR) before sequencing to identify read-through variants and avoid interference from the PMS2CL pseudogene. This is a highly customized and resource-intensive approach to the analysis of a single gene in every sample. Having developed an approach that maximizes the use of our established workflows and capabilities, we are able to offer sequencing of this difficult but important region of PMS2 while maintaining our commitment to affordability.

Invitae's approach to PMS2

Invitae’s approach to the evaluation of exons 12–15 of PMS2 is a two-step process for read-through variants and a three-step process for deletions and duplications (Figure 1). The first step for both types of variants is a bioinformatics screen in which sequence reads derived from both PMS2 and the paralogous PMS2CL gene are analyzed for the presence of variants using PMS2 as the reference sequence. For read-through variants, non-benign variants identified in the screen are definitively assigned to PMS2 or PMS2CL using Sanger sequencing of LR-PCR products of PMS2 (exons 12–15) and PMS2CL (exons 3–6). For deletion/duplication variants, the second step is to confirm the bioinformatics screen call with MLPA, and to account for the possibility of gene conversion, a final step with LR-PCR is used to disambiguate the location of the variant.6

Figure 1. Invitae’s method of PMS2 sequencing and deletion/duplication analysis

Validation Data

This approach was validated with samples known to have specific variants in these exons for both genes (reference set). For validation of the read-through method, we analyzed 32 unique samples carrying 205 true positive and 34,876 true negative variants in PMS2 or PMS2CL and demonstrated an accuracy, reproducibility, and analytical sensitivity and specificity of 100% (Table 1). For validation of the deletion/duplication method, we analyzed 28 unique samples carrying 90 true positive and 50 true negative individual exon variants in PMS2 or PMS2CL and demonstrated an accuracy, reproducibility, and analytical sensitivity and specificity of 100% (Table 2).

Table 2 and Table 3. Analytical sensitivity and specificity of PMS2 sequencing and deletion/duplication analysis


1. Lynch, HT, et al. Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications. Clinical Genetics. 2009; 76(1):1-18. PMID: 19659756
2. Gill, S, et al. Isolated loss of PMS2 expression in colorectal cancers: frequency, patient age, and familial aggregation. Clinical Cancer Research. 2005; 11:6466-6471. PMID: 16166421
3. Halvarsson, B, et al. The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer. Familial Cancer. 2006; 5:353-358. PMID: 16817031
4. Truninger, K, et al. Immunohistochemical analysis reveals high frequency of PMS2 defects in colorectal cancer. Gastroenterology. 2005;128:1160-1171. PMID: 15887099
5. Hayward, BE, et al. Extensive gene conversion at the PMS2 DNA mismatch repair locus. Human Mutation. 2007; 28(5):424-30. PMID: 17253626
6. Vaughn CP, et al. Avoidance of pseudogene interference in the detection of 3’ deletions in PMS2. Human Mutation. 2011; 32(9):1063-71. PMID: 21618646

To learn more, please read our PMS2 sequencing and deletion/duplication validation statement.

Clinical evaluation of multi-gene hereditary cancer panels

To demonstrate the value of multi-gene panels in hereditary cancer risk assessment, Invitae collaborated with Stanford University researchers, James Ford, M.D. and Allison W. Kurian, MD, MSc. The results of this research, recently published in the Journal of Clinical Oncology, show that that multi-gene hereditary cancer panels can offer comparable performance to traditional BRCA1/2 genetic testing and can provide additional clinical benefit to doctors and patients seeking cancer risk assessment.

To learn more about this publication, visit our Clinical Studies page.

Invitae’s approach to testing SMN1 and SMN2 for spinal muscular atrophy

Complete loss of SMN1 gene function results in spinal muscular atrophy (SMA), an early-onset debilitating neuromuscular disorder characterized by loss of motor neurons in the spinal cord. SMN1 has a near-identical gene copy named SMN2 also located on chromosome 5, approximately 800 kilobases from SMN1. The coding regions of SMN2 and SMN1 differ from one another by a single nucleotide in exon 7*, which we term the gene-determining variant (GDV). This difference adversely affects splicing of the exon and leads to very little full length protein production from the SMN2 gene.

The majority of pathogenic changes in SMA are deletions of SMN1 or gene conversion of SMN1 to SMN2. In addition, rare inactivating sequence variants can occur in SMN1. About 95%–98% of individuals with SMA have zero copies of SMN1 and about 2%–5% are compound heterozygotes, with a deletion of SMN1 on one chromosome and a pathogenic sequence variant in SMN1 on the other chromosome. Notably, the number of SMN2 copies is highly variable among individuals. This number influences the SMA phenotype in patients with SMN1 loss, with severity decreasing and age of onset increasing as the number of SMN2 copies increases.1,2

Challenges in SMA testing and Invitae's NGS-based approach

Most laboratories traditionally diagnose SMA by performing multiplex ligation-dependent probe amplification (MLPA) or quantitative PCR (qPCR) to identify loss of SMN1 exon 7*. These approaches have significant technical limitations and are difficult to efficiently integrate into broader testing.

To address these limitations we developed a comprehensive next-generation sequencing (NGS)-based approach with a customized bioinformatics solution to offer simultaneous sequencing and copy number analysis of these difficult genes while maintaining our commitment to quality and affordability.

Table 1

NGS-based methodology

Invitae has developed a sophisticated assay and bioinformatics solution to accurately detect pathogenic changes in SMN1 and determine SMN2 copy number. First, we align sequencing reads derived from both SMN1 and SMN2 to an SMN1 reference sequence. We then measure total SMN1 + SMN2 copy number using a modified version of CNVitae, our custom-built copy number variant detection algorithm that utilizes NGS read counts. Once we have the total SMN1/2 copy number, individual SMN1 and SMN2 exon 7* copy numbers are determined using the exon 7* GDV. This simultaneous determination of SMN1 and SMN2 exon 7* copy numbers enables high confidence calls for both SMN1 and SMN2** (Figure 1).

We also use the exon 7* GDV to unambiguously place sequence variants in exon 7* of SMN1 and SMN2. The remaining exons (1–6) of SMN1 and SMN2 are identical in sequence, and therefore while we can accurately identify sequence and copy number variants in these exons, their true location within SMN1 or SMN2 cannot be determined. Even though disambiguation is not possible for variants in exons 1–6, their identification can inform the diagnosis of rare compound heterozygous affected individuals.

SMN1/2 exon 7* copy number variants are confirmed by ligation-dependent sequencing, an Invitae innovation that transforms traditional MLPA into a highly scalable NGS method. Sequence variants in exon 7* are confirmed using single-molecule PacBio sequencing, which enables the phasing of the variant with the GDV to unambiguously place the variant in either SMN1 or SMN2.

Figure 1: SMN1/2 bioinformatics method

Reads derived from both SMN1 and SMN2 are aligned to SMN1, and combined SMN1/2 copy number is determined using Invitae’s read count-based copy number variant detection algorithm, CNVitae. SMN1- and SMN2-specific exon 7* copy number is resolved by counting reads with the gene determining variant in exon 7*.

Figure 1: SMN1/2 bioinformatics method


Our SMN1/2 approach was validated on a set of nine samples available from an external commercial repository of biological samples. SMN1 exon 7* copy number information was previously determined through traditional methods, and SMN2 copy number was known for a subset of these samples.3 Our method showed 100% sensitivity and specificity for SMN1 and SMN2 copy number, and notably its higher resolution for determining SMN2 copy number enabled us to obtain accurate results for three samples for which copy number had been imprecisely determined with traditional methods previously.3

SMN1 and SMN2 population frequency

Table 2


*Reference sequence NM_000344.3, which is used to describe SMN1 sequence variants, contains 8 protein-coding exons. Due to historical reasons, the second and third exons are conventionally referred to as exons 2a and 2b, and the subsequent exons are referred to as exons 3–7 (PMID: 8838816). At Invitae, systematic exon numbering is used for all genes, including SMN1 and SMN2. For this reason, the gene-differentiating exon conventionally referred to as exon 7 in the literature and in this whitepaper is referred to as exon 8 in our clinical reports.
**Copy number of SMN2 exon 7* is expected to represent copy number for the entire SMN2 gene, and will only be reported for individuals with a positive result in SMN1. CNVs limited to exons 1–6 of SMN1 or SMN2 will not be reported. This assay cannot detect silent carriers (individuals that have 2 functional copies of SMN1 on one chromosome and zero copies on the other). Therefore a negative result for carrier testing greatly reduces but does not eliminate the chance that a person is a carrier.


1. Mailman MD et al. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet. Med. 2002;4:20–6. PMID: 11839954
2. Swoboda KJ et al. Natural history of denervation in SMA: relation to age, SMN2 copy number, and function. Ann Neurol. 2005;57:704– 12. PMID: 15852397
3. Stabley DL et al. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR. Molecular Genetics & Genomic Medicine 2015;3(4):248- 257. PMID: 26247043
4. Hendrickson BC et al. Di erences in SMN1 allele frequencies among ethnic groups within North America. Journal of Medical Genetics 2009;46:641-644. PMID: 19625283

Download the Invitae’s approach to testing SMN1 and SMN2 for spinal muscular atrophy one-page PDF of this white paper, which includes an appendix not shown here.

We are happy to share more details on any of our validation studies with you. Please contact Client Services to request additional information.