Combinatorial batching of DNA for ultralow-cost detection of pathogenic variants

We demonstrate that the DoBSeq approach to mass genetic screening for rare disease-causing variants is a reliable and cost-effective method, which is likely to be highly scalable and hence applicable to population screening. The high performance and ability to directly pinpoint carriers of genetic variants are a product of the repeated sequencing of the same individual in two separate batches. Essentially, our results fully support that DoBSeq applied on large cohorts can directly identify single individuals with rare variants. The identified carriers may then be confirmed using conventional sequencing, after which clinical reporting could take place in adherence to existing best practices [18, 19]. To eliminate interpretation biases in the present analysis, we purposefully excluded variants that were internally classified as pathogenic (Fig. 2).

Surprisingly, our data showed that false positive LoF/P variants with passable quality parameters were widespread when the sequencing results from stand-alone batches were assessed in isolation (Fig. 1A–B, D–E). However, by filtering variants to those seen in a combination of one column and one row batch, i.e., appearing twice in two different batches, we could clearly distinguish between true and false positive variants. This suggests that, using our method, single batched sequencing (in which carriers cannot be pinpointed) would require copious individual resequencing. Considering the high number of RDs in the general population, the pursuit of just the true positive variants could become costly if dozens or even hundreds of genes were screened simultaneously, with each finding requiring resequencing of all individuals in the stand-alone batch. Based on our data, false positives in stand-alone batches were common, however, our design was not specifically optimized with highly accurate variant calling in stand-alone batches in mind, hence the resequencing needed in pursuit of variant which would ultimately turn out to be false, could not be reliably assessed based on our data.

Currently, well over 400 gene-disease pairs are considered highly actionable in childhood with respect to age-of-onset and/or timing of intervention, and at least an additional 25 are highly actionable in adulthood [5]. Each of these conditions may be considered viable candidates for population screening, yet, primarily due to cost restrictions, few are routinely screened for in any healthcare system and no screening currently uses genomics up-front [8, 20].

A recent simulation model evaluated the cost-effectiveness of universal screening for a panel including 11 pediatric CPS genes [16], which fully overlapped with the 113-gene panel used in our study. Because RDs, such as pediatric CPSs, by definition have a very low prevalence, the number-needed-to-diagnose (NND) in an unselected cohort is high. For individual rare conditions, such as WT1-related disorders, the price per diagnosis (PPD) at the estimated cost of doing a single genetic test likely exceeds $10 million (Fig. 3C). Of course, by using panels, several conditions can be screened for simultaneously, lowering the NND for any one condition, yet, for the 11 pediatric CPS genes, Yeh et al. [16] conclude that at the current best price per sample (PPS) of $55, tumor surveillance strategies are cost-prohibitive ($244,860 per life-year gained). However, at a PPS of $20, surveillance approaches liberal cost-effectiveness (<$100,000 per life-year gained) (Fig. 3D). Practically, the PPD must be added to the isolated cost of the tumor surveillance strategy, making it increasingly likely that any given treatment, which is cost-effective in isolation, will remain cost-effective as PPD is reduced. According to the models developed by Yeh et al., the tumor surveillance strategies and treatments available for the 11 conditions they studied will be even conservatively cost-effective (<$50,000 per life-year gained) if PPS drops below $8, which we demonstrate to be highly possible when DoBSeq is run at scale (Fig. 3D).

DoBSeq has the potential to advance genetically based risk stratification, and the cost-effectiveness of doing so, for hundreds of actionable RDs caused by rare genetic variants. This is by no means limited to DNA samples from neonatal blood spots. Several studies have investigated the cost-effectiveness of population screening for adult CPSs [21,22,23,24,25], and one such study [25], investigating the impact of PPS on universal adult screening for BRCA1/2 and MMR genes, found it to be conservatively cost-effective even at PPS exceeding $1000. Lowering PPS to those estimated for DoBSeq running at scale could lead to prices per life-year gained that approach cost-saving (Additional file 1: Fig. S11, Fig. S12). Most adult CPSs are believed to be undiagnosed, and therefore population-based cancer screening stands to improve with genetically informed precision prevention [24].

The promising aspects of DoBSeq must be viewed in light of some limitations of the method and of this study. If multiple individuals in the same DoBSeq matrix carry exactly the same variant, it may not be possible to pinpoint the carriers directly. In this event, however, the number of possible carriers will be limited to a small group amenable for individual (re)sequencing. For instance, if two individuals carried the same variant, (re)sequencing of four individuals would be required to identify the two true carriers. Still, genetic heterogeneity, meaning that a specific disease may be caused by a myriad of distinct genetic defects, generally makes multiple carriers of molecularly identical variants in the same matrix astronomically unlikely. This was also found in our study, e.g., for TP53 where four distinct variants all caused the same condition (Li-Fraumeni Syndrome) associated with a high risk of childhood cancers [26] (Fig. 2).

In some populations, pathogenic founder variants causing genetic diseases that are rare globally may be common enough to make person-specific variant detection using DoBSeq challenging. Nevertheless, founder variants are virtually always known and well-studied in the populations that carry them at high frequencies, and therefore such variants may be better suited for bespoke detection methods, such as was done for the Brazilian TP53 founder variant, which was neonatally, and highly cost-effectively, screened for using a direct restriction fragment length polymorphism assay in the Paraná region of Brazil [27]. Conversely, DoBSeq’s use lies in identifying the highly heterogeneous mutational spectrum of non-founder, i.e., personal, variants, which cause the vast majority of serious genetic diseases in humans. Theoretically, the presented method should provide conclusive results for virtually all loci where variants have an allele frequency of 0.001% or lower (Additional file 1: Table S3).

Another possible limitation was that prior to batching, our method harmonized DNA concentration across all samples, which added a minor cost for each sample. At scale, this may impact overall cost-effectiveness estimates and we did not test whether removing this step impacted the performance of DoBSeq. Lastly, structural variants, known to cause around 10% of RD [28], were not investigated as part of the present study.

Lately, combinatorial pooling strategies, like DoBSeq, have received increased attention as a theoretical alternative to the extensive and costly population screening for the SARS-CoV-2 virus [29]. While the methods appear similar, a crucial difference is that a heritable disease may be caused by a myriad of distinct genetic variants, whereas SARS-CoV-2 tests, by and large, are positive in the same way.

Finally, we would be remiss not to mention that the implementation of mass genetic screening of healthy neonates, children, and/or adults, precipitates critical ethical considerations [30]. Ethics is not a focus of our current study, however, it is important to note that our method detects rare variants only, thus limiting the amount of personal genetic data obtained per individual. Moreover, if our method is developed further, the low price per sample may extend accessibility in low- and middle-income countries, making it more equitable than current individual NGS-based methods.

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