Low‐pass whole genome sequencing is a reliable and cost‐effective approach for copy number variant analysis in the clinical setting

Abstract

IntroductionNext generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low‐pass whole genome sequencing (LP‐WGS). Here, we evaluated the performance of LP‐WGS to detect copy number variants (CNVs) in clinical cytogenetics.Materials and MethodsDNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75 kb to 90.3 Mb, including aneuploidies and two cases of mosaicism.ResultsAll CNVs were successfully detected by LP‐WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP‐WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP‐WGS approach are at least similar to those provided by CMA.DiscussionOur data show the potential use of LP‐WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP‐WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

Low-pass whole genome sequencing as a cost-effective alternative to chromosomal microarray analysis for low- and middle-income countries

“…In this study, we applied LP-WGS in a large multicentric Brazilian cohort in order to evaluate the costeffectiveness of this approach as a diagnostic alternative to replace CMA and provide more equal access to genetic testing in our country. Notably, the diagnostic yield (P/LP CNVs) in this cohort (16%) is comparable to the 15-20% determined for CMA and the CNV detection rate is highly e cient, costing only a quarter of CMA 14 . Regarding the frequency of VUS, it is estimated in approximately 6% from postnatal microarrays 32 , similar to our ndings with LP-WGS.…”

“…In our previous work, we showed that LP-WGS, also known as shallow sequencing, is equally sensitive and accurate as CMA in detecting copy number changes both in prenatal and postnatal samples 14 . LP-WGS is already widely applied in multiple international diagnostic centers for CNV detection in a variety of clinical applications, including NIPT 27 , pre-implantation genetic testing 28 , liquid biopsy 29 , and solid tumor analysis 30 .…”

“…A cost-effective alternative method to detect CNVs with similar resolution to CMA is the low-pass whole genome sequencing (LP-WGS), a high-throughput sequencing of the entire human genome at low-coverage [9][10][11][12][13] . In a recent publication, we demonstrated that LP-WGS at an average depth of 1x is as sensitive and speci c to detect microscopic and submicroscopic chromosome alterations as CMA 14 . LP-WGS detects CNVs at least 50 kb large and has a coverage suitable to detect the well-recognized microdeletion and microduplication syndromes, with an estimated cost of one quarter compared to CMA 14 .…”

“…In a recent publication, we demonstrated that LP-WGS at an average depth of 1x is as sensitive and speci c to detect microscopic and submicroscopic chromosome alterations as CMA 14 . LP-WGS detects CNVs at least 50 kb large and has a coverage suitable to detect the well-recognized microdeletion and microduplication syndromes, with an estimated cost of one quarter compared to CMA 14 . Therefore, this study was designed to evaluate, in a large cohort of patients from Nonpro t Organizations and University Centers, the e ciency and reliability of LP-WGS as a more affordable diagnostic option to replace CMA.…”

“…To identify CNVs, we used the SNP-FASST2 segmentation algorithm, based on the Hidden Markov Model, with a sensitivity threshold of 1.0 E-6. A genomic segment was considered duplicated or deleted when the log 2 ratio of a given region encompassing at least three targets was above 0.3 or below − 0.3, respectively 14,17,18 . Detected CNVs were classi ed according to their potential clinical impact as Pathogenic (P), Likely Pathogenic (LP), Variants of Uncertain Signi cance (VUS), Likely Benign (LB) and Benign (B), according to the European guidelines for constitutional cytogenomics analysis, American College of Medical Genetics (ACMG) and Clinical Genome Resource (ClinGen) guidelines 19,20 .…”