Summary

Because Orchid Preimplantation Genetic Testing with Whole-Genome Sequencing (PGT-WGS) generates whole-genome sequencing data for embryos, some IVF patients choose to export the raw data and use commercial genetic data upload services for secondary analysis to gain additional information about traits or complex conditions.

Orchid PGT-WGS provides the most data for secondary data analysis compared to other PGT options. However, secondary analysis of PGT data has limitations which are important to understand before making plans, and different types of secondary analysis have different risks of inaccurate results

Orchid does not endorse or give any guidance on how to interpret the results of secondary embryo data analysis, and cannot vet the accuracy of specific analysis services.  However, secondary analysis of embryo data is becoming increasingly common due to new commercial offerings.  This guide helps patients understand their options and limitations when navigating this process.

Secondary analysis after Orchid PGT-WGS

Secondary analysis of PGT data happens after your clinical report has been delivered and you have discussed your results with an Orchid genetic counselor.  Upon patient request, Orchid releases raw sequencing data in FASTQ format for euploid embryos analyzed via PGT-WGS monogenic screening or Genetic Risk Scores.

These raw data files can be uploaded to several secondary data analysis services which provide exploratory reports for diseases or traits for which Orchid does not deliver a clinical report.   Because Orchid generates Whole Genome Sequencing (WGS) data, most forms of secondary analysis are technically possible, including custom GRS, traits, and additional monogenic analysis, which patients may use when deciding which embryo to transfer.  However, important limitations apply and are discussed later.  

Depending on the service used, your doctor may or may not receive an additional report with this information.  However, it is always recommended that you discuss tradeoffs with your IVF physician when prioritizing embryos for transfer. Not all IVF physicians will allow the use of non-clinical data/reports when selecting an embryo for transfer. 

Secondary analysis after non-Orchid PGT

While every lab has a different process for requesting raw data from PGT tests, the basic flow operates similarly to Orchid — data is only released after the clinical PGT report has been delivered and the patient explicitly requests and consents to access the raw data.

Some PGT labs do not release raw data in a format usable for secondary genetic analysis. . However, some other PGT labs release raw data for patient embryos in microarray or FASTQ format.  Orchid is currently the only commercial PGT provider offering clinical whole-genome sequencing of embryos at >30x depth (each letter on the embryo's genome is read about 30 times); data released by other labs in FASTQ format may represent low depth sequencing data of < 1x depth; microarray data, on the other hand, sequences specific sites at high depth, but sequences < 1% of the genome.

Low-depth or microarray data cannot be used for all forms of secondary analysis, and all forms of analysis are subject to greater limitations than when using whole-genome data. Custom GRS or exploratory traits may be possible. However, secondary analysis must use genetic imputation to fill in the “missing pieces” in the raw data using public biobanks at reduced accuracy, especially for populations not of European ancestry who are not not well-represented in biobanks.  This reduces the precision of GRS for polygenic diseases where risk is determined in large part by a small number of genetic variants (for example, APOE sites and Alzheimer’s disease)

However, accurate monogenic panel screening on microarray or low-depth WGS data is not possible because the vast majority of the embryo’s genome has not been sequenced at high enough depth for variant-level analysis.

Challenges during secondary analysis

When sequencing data is analyzed by a different lab than the lab that originally produced the sequencing data, there is an elevated risk of inaccurate results.  Before planning a secondary analysis of embryo data, it’s important to understand the limitations.

Limitations on genomic region and variants

While the data from different genetic sequencing laboratories looks similar — it is usually exported either in common FASTQ or VCF formats — the genetic data generated by different sequencing technologies has specific limitations.  In particular, the DNA amplification used to take DNA from a ~5 cell biopsy and use it for WGS sequencing will produce data which is more accurate in some locations on the genome, and for different variant types, than others.  Additionally, the type of sequencing technology impacts variant detection accuracy.

Before adding genes to our monogenic screening panels, Orchid performs a thorough validation process to determine that those genes have good coverage and have low rates of both false positive and false negative variant findings on PGT-WGS embryo genetic data. Orchid’s panels currently cover 1200 genes related to serious health conditions, while the whole human genome contains over ~20,000 protein-coding genes, many of which are not yet well-understood.  Additionally, to ensure accuracy, certain genes (such as those with pseudogenes) or variant types (such as small insertions in homopolymer regions) are not included in our screening. Orchid also does not report changes in genes that have an uncertain relationship to disease (Variants of Uncertain Significance, or VUS) or carrier status for recessive conditions. The methods used to produce sequencing data, and the limitations of Orchid’s screening, are listed at the bottom of the PGT-WGS report your physician received.

When secondary analysis is performed by a third party lab, these limitations may not be accounted for during analysis.  If this happens, the analysis may report significant numbers of false positive findings — variants which are reported by the analysis pipeline, but do not represent real genetic variants in an embryo.

No confirmatory testing of variants

When Orchid screens for a specific variant ordered by your physician based on personal/family history (PGT-M), or detects a variant during monogenic panel screening, several types of confirmatory testing are performed after WGS analysis on your embryos to confirm that the variant was neither a false positive (incorrectly detected) or false negative (incorrectly missed).

Orchid is typically able to go back and use the embryo DNA to perform additional confirmatory testing, such as Sanger sequencing (which checks for a specific variant), because our lab performed the original Whole Genome Sequencing.  A lab which analyzes only exported data cannot perform confirmatory testing because they do not have the equipment or DNA to perform followup genetic testing.   In this situation, a re-biopsy may be the only option for further analysis, which may lower pregnancy success rates.  This limitation results in elevated rates of false positive findings.

Risks incurred by different forms of secondary analysis

These limitations affect the accuracy of different forms of secondary analysis in different ways.  In general, the limitations inherent to secondary analysis introduce the least accuracy risk when generating Genetic Risk Scores, and introduce the largest accuracy risks when performing monogenic panel screening.

Genetic Risk Scores

Highly polygenic GRS combine results across many genetic variants and are less sensitive to false positive or false negative calls at a single location.  Additionally, GRS usually analyze only common variants, which are at lower risk of false positive detection, and common missing variants can be “filled in” using genetic imputation tools.  However, GRS for certain diseases depend heavily on a small number of discrete variants (“Oligogenic”); for example, APOE status is the primary driver of Alzheimer's risk, and these scores are more sensitive to misaligned analysis.

It is important to distinguish the accuracy limitations introduced during a secondary analysis from the predictive value of the Genetic Risk Score itself.  Even in situations where a GRS can be accurately computed on embryo data during a secondary analysis, it may have low predictive value — for instance, a person at 98th percentile of risk may have only a 1.4x average disease risk, and not be highly meaningful in predicting absolute risk.

Orchid’s PGT-WGS report includes only scores which are both predictive and meaningfully stratify risk — risk is only considered elevated if the risk is at least 2x the population average risk — and can be accurately computed on embryo data.  Genetic Risk Scores produced during a secondary analysis may be accurate and predictive, or may have lower standards for predictiveness and accuracy.

Analysis for specific variants

In most cases, secondary analysis which looks for a specific genetic variant (for example, a variant a parent read an article about after embryo screening had already been performed) is at lower risk of false positive findings, because the odds of a false positive occurring at a specific familial site is generally low.

However, the risk of a false negative finding is elevated if the variant is in a region not suited to monogenic screening, and due to the lack of confirmatory testing in a data-only analysis.  As a result, secondary analysis should not be used in lieu of PGT-M on variants linked to serious health conditions.  If a harmful variant is detected in a parent after Orchid’s PGT-WGS is complete, in many cases Orchid is able to perform a reanalysis which includes PGT-M for the target variant by using the data and DNA from the original test.  An Orchid genetic counselor can help you start that process.

Monogenic panel screening

Secondary analysis which performs general monogenic panel screening is at the highest risk of inaccurate results.  If panel screening is performed across the entire genome, or on a large number of genes, without regards to coverage limitations, false positive variants will be detected on every embryo.  

False positive variants detected during WGS analysis make it extremely difficult for parents and physicians to decide which embryo to transfer. If the variant was identified in a region not amenable to monogenic screening, additional sequencing cannot be performed to check if a variant is “real”. Even if access to the original biopsy is available, embryo DNA is limited, and targeted sequencing cannot be performed on a large number of variants simultaneously. As a result, Orchid PGT-WGS patients are advised against performing secondary panel screening using their embryo data.

What to understand before ordering a secondary analysis

Before engaging a third party service to perform a secondary analysis of your embryo data, it is important to understand the lab’s methodology and tests as well as what your options will be in the event of an unexpected finding:

• If you hope to use embryo data for secondary analysis, it’s important to make sure your PGT lab produces data compatible with the analysis you wish to perform later.  
• If using Orchid data, make sure the service has read the methods at the bottom of your PGT-WGS report, and confirm that their data pipeline is validated on the sequencing technology that was used by Orchid.
• Confirm that this analysis accounts for the limitations on the types of genetic variants screened.  This information is also found in the limitations of your PGT-WGS report.

• If receiving Genetic Risk Scores or traits, confirm that the lab uses genetic imputation tools to address variants in regions of low coverage.
• Orchid will not be able to provide counseling on findings from secondary analysis 

Most importantly, before performing a secondary analysis, you and your partner should plan what you will do if an embryo screens positive for a condition and understand how/if your IVF center may utilize these new results for decisions regarding embryo transfer.  In most cases, there will be no way to definitively rule out a false positive secondary finding.

Learn more about Orchid Whole-Genome Embryo Screening

This guide is broad information to inform patients about their options and the inherent limitations of secondary embryo data analysis.  Please discuss any plans with your physician before making decisions.

If you’d like to learn more about embryo screening, or get started, you can schedule a call now with one of our genetic counselors.