Detection of Copy Number Alterations by Shallow Whole-Genome Sequencing of Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

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.

PREFACE: In silico pipeline for accurate cell-free fetal DNA fraction prediction.

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 (nfemale = 2468, nmale = 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.

WisecondorX: improved copy number detection for routine shallow whole-genome sequencing.

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.

Implementation of non-invasive prenatal testing by semiconductor sequencing in a genetic laboratory.

OBJECTIVES: To implement non-invasive prenatal testing (NIPT) for fetal aneuploidies with semiconductor sequencing in an academic cytogenomic laboratory and to evaluate the first 15-month experience on clinical samples. METHODS: We validated a NIPT protocol for cell-free fetal DNA sequencing from maternal plasma for the detection of trisomy 13, 18 and 21 on a semiconductor sequencing instrument. Fetal DNA fraction calculation for all samples and several quality parameters were implemented in the workflow. One thousand eighty-one clinical NIPT samples were analysed, following the described protocol. RESULTS: Non-invasive prenatal testing was successfully implemented and validated on 201 normal and 74 aneuploid samples. From 1081 clinical samples, 17 samples showed an abnormal result: 14 trisomy 21 samples, one trisomy 18 and one trisomy 16 were detected. Also a maternal copy number variation on chromosome 13 was observed, which could potentially lead to a false positive trisomy 13 result. One sex discordant result was reported, possibly attributable to a vanishing twin. Moreover, our combined fetal fraction calculation enabled a more reliable risk estimate for trisomy 13, 18 and 21. CONCLUSIONS: Non-invasive prenatal testing for trisomy 21, 18 and 13 has a very high specificity and sensitivity. Because of several biological phenomena, diagnostic invasive confirmation of abnormal results remains required. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.