Shallow whole-genome sequencing to infer copy number alterations (CNAs) in the human genome is rapidly becoming the method par excellence for routine diagnostic use. Numerous tools exist to deduce aberrations from massive parallel sequencing data, yet most are optimized for research and often fail to redeem paramount needs in a clinical setting. Optimally, a read depth-based analytical software should be able to deal with single-end and low-coverage data—this to make sequencing costs feasible. Other important factors include runtime, applicability to a variety of analyses and overall performance. We compared the most important aspect, being normalization, across six different CNA tools, selected for their assumed ability to satisfy the latter needs. In conclusion, WISECONDOR, which uses a within-sample normalization technique, undoubtedly produced the best results concerning variance, distributional assumptions and basic ability to detect true variations. Nonetheless, as is the case with every tool, WISECONDOR has limitations, which arise through its exclusiveness for non-invasive prenatal testing. Therefore, this work presents WisecondorX in addition, an improved WISECONDOR that enables its use for varying types of applications. WisecondorX is freely available at https://github.com/CenterForMedicalGeneticsGhent/WisecondorX.

Background: Accurate lung cancer classification is crucial to guide therapeutic decisions. However, histological subtyping by pathologists requires tumor tissue—a necessity that is often intrinsically associated with procedural difficulties. The analysis of circulating tumor DNA present in minimal-invasive blood samples, referred to as liquid biopsies, could therefore emerge as an attractive alternative. Methods: Concerning adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, our proof of concept study investigates the potential of liquid biopsy-derived copy number alterations, derived from single-end shallow whole-genome sequencing (coverage 0.1–0.5×), across 51 advanced stage lung cancer patients. Results: Genomic abnormality testing reveals anomalies in 86.3% of the liquid biopsies (16/20 for adenocarcinoma, 13/16 for squamous cell, and 15/15 for small cell carcinoma). We demonstrate that copy number profiles from formalin-fixed paraffin-embedded tumor biopsies are well represented by their liquid equivalent. This is especially valid within the small cell carcinoma group, where paired profiles have an average Pearson correlation of 0.86 (95% CI 0.79–0.93). A predictive model trained with public data, derived from 843 tissue biopsies, shows that liquid biopsies exhibit multiple deviations that reflect histological classification. Most notably, distinguishing small from non-small cell lung cancer is characterized by an area under the curve of 0.98 during receiver operating characteristic analysis. Additionally, we investigated how deeper paired-end sequencing, which will eventually become feasible for routine diagnosis, empowers tumor read enrichment by insert size filtering: for all of the 29 resequenced liquid biopsies, the tumor fraction could be increased in silico, thereby “rescuing” three out of five cases with previously undetectable alterations. Conclusions: Copy number profiling of cell-free DNA enables histological classification. Since shallow whole-genome sequencing is inexpensive and often fully operational at routine molecular laboratories, this finding has current diagnostic potential, especially for patients with lesions that are difficult to reach.

Methylation profiling of circulating cell-free DNA (cfDNA) is of great interest as a liquid biopsy assay for the detection and monitoring of cancer and other pathologies. Here we describe ‘circulating cell-free DNA reduced representation bisulfite sequencing’ (cf-RRBS), enabling the use of highly effective RRBS on fragmented plasma cfDNA. This method enriches the CpG-rich RRBS target regions by enzymatic degradation of all off-target DNA rather than by targeted capture, in contrast to previous methods. Critical steps are fully enzymatic in a single-tube, making it rapid, cost-effective, robust, and easily implemented on a liquid-handling station for high-throughput sample preparation. We benchmark cf-RRBS results to those obtained by previous more complex methods and exemplify its use for accurate non-invasive subtyping of lung cancer, a frequent onco-pathology task. cf-RRBS enables any molecular pathology lab to tap into the cfDNA methylome, only making use of off-the-shelf reagents and open-source data analysis tools. One Sentence Summary: Novel methodology enables facile and cost-effective methylation profiling of fragmented plasma DNA, allowing for routine liquid biopsy clinical diagnostics, exemplified here for differential diagnosis of lung cancer subtypes.

Objective: During routine noninvasive prenatal testing (NIPT), cell‐free fetal DNA fraction is ideally derived from shallow‐depth whole‐genome sequencing data, preventing the need for additional experimental assays. The fraction of aligned reads to chromosome Y enables proper quantification for male fetuses, unlike for females, where advanced predictive procedures are required. This study introduces PREdict FetAl ComponEnt (PREFACE), a novel bioinformatics pipeline to establish fetal fraction in a gender‐independent manner.Methods: PREFACE combines the strengths of principal component analysis and neural networks to model copy number profiles.Results: For sets of roughly 1100 male NIPT samples, a cross‐validated Pearson correlation of 0.9 between predictions and fetal fractions according to Y chromosomal read counts was noted. PREFACE enables training with both male and unlabeled female fetuses. Using our complete cohort (n_female = 2468, n_male = 2723), the correlation metric reached 0.94.Conclusions: Allowing individual institutions to generate optimized models sidelines between‐laboratory bias, as PREFACE enables user‐friendly training with a limited amount of retrospective data. In addition, our software provides the fetal fraction based on the copy number state of chromosome X. We show that these measures can predict mixed multiple pregnancies, sex chromosomal aneuploidies, and the source of observed aberrations.

Context.—: In routine clinical practice, tumor tissue is stored in formalin-fixed, paraffin-embedded blocks. However, the use of formalin-fixed, paraffin-embedded tissue for genome analysis is challenged by poorer DNA quality and quantity. Although several studies have reported genome-wide massive parallel sequencing applied on formalin-fixed, paraffin-embedded samples for mutation analysis, copy number analysis is not yet commonly performed. Objective.—: To evaluate the use of formalin-fixed, paraffin-embedded tissue for copy number alteration detection using shallow whole-genome sequencing, more generally referred to as copy number variation sequencing. Design.—: We selected samples from 21 patients, covering a range of different tumor entities. The performance of copy number detection was compared across 3 setups: array comparative genomic hybridization in combination with fresh material; copy number variation sequencing on fresh material; and copy number variation sequencing on formalin-fixed, paraffin-embedded material. Results.—: Very similar copy number profiles between paired samples were obtained. Although formalin-fixed, paraffin-embedded profiles often displayed more noise, detected copy numbers seemed equally reliable if the tumor fraction was at least 20%. Conclusions.—: Copy number variation sequencing of formalin-fixed, paraffin-embedded material represents a trustworthy method. It is very likely that copy number variation sequencing of routinely obtained biopsy material will become important for individual patient care and research. Moreover, the basic technology needed for copy number variation sequencing is present in most molecular diagnostics laboratories.