Next-generation Sequencing Data Analysis Research Articles

REVERSE: a user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments.

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single-nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that implements an integrated computational pipeline through a graphical user interface, which performs both pre-processing and detailed sequence level analyses within minutes. Raw FASTQ files are quality-filtered, dereplicated, and trimmed before being analyzed by either of two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and user-defined sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice and generates a consensus sequence. Both pipelines generate downloadable spreadsheets and high-resolution figures. Collectively, REVERSE is a one-stop solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.

Engineering of Src Homology 2 Domain Leading to Sulfotyrosine Recognition With a High Affinity by Integrating a Distinctive Selection Theme and Next-Generation Sequencing.

Tyrosine sulfation plays a vital role in various biochemical reactions. Although sulfated tyrosine (sTyr) has a similar structure to phosphotyrosine (pTyr), the number of available sTyr sites is significantly less than that of pTyr sites, mainly because of the lack of effective sTyr probes. A few sTyr binders were identified on the basis of structural similarity by engineering the pTyr-binding pocket of an Src Homology 2 (SH2) domain through phage selections against sTyr peptides. Nevertheless, they still interact with pTyr peptides with comparable affinity. This study aims to identify sTyr superbinders using the SH2 domain as a template. We created a distinctive phage selection scheme that separately covered selections against sTyr and pTyr peptides, followed by next-generation sequencing (NGS). After selections, phage pools showed strong enzyme-linked immunosorbent assay (ELISA) signal intensities for both modified peptides, indicating that the variants evolved with a high affinity for these peptides, which causes difficulty in identifying sTyr-specific binders. In contrast, NGS data from selected pools showed significant differences, suggesting the enrichment of sTyr-specific variants during selections. Accordingly, we obtained the sTyr features based on NGS data analysis and prioritized a few potential sTyr binders. The variant SH2-4 showed a stronger affinity for sTyr than pTyr and was superior to previous sTyr binders as measured by the Biolayer Interferometry assay. In summary, we described the strategy of integrating NGS data mining with a novel selection scheme to identify sTyr superbinders.

Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database

The increasing number and diversity of genetically modified organisms (GMOs) for the food and feed market calls for the development of advanced methods for their detection and identification. This issue can be addressed by next generation sequencing (NGS). However, the efficiency of NGS-based strategies depends on the availability of bioinformatic methods to find sequences of the transgenic insert and junction regions, which is a challenging topic. To facilitate this task, we have developed Nexplorer, a sequence-based database in which annotated sequences of GM events are stored in a structured, searchable and extractable format. As a proof of concept, we have developed a methodology for the analysis of sequencing data of DNA walking libraries of samples containing GMOs using the database. The efficiency of the method has been tested on datasets representing various scenarios that can be encountered in routine GMO analysis. Database-guided analysis allowed obtaining detailed and reliable information with limited hands-on time. As the database allows for efficient analysis of NGS data, it paves the way for the use of NGS sequencing technology to aid routine detection and identification of GMO.

Molecular characterization of renal cell carcinoma with venous tumor thrombus: Genomic aberrations with renal vein wall invasion.

385 Background: Renal cell carcinoma (RCC) patients with tumor thrombus (TT) have been observed distinct phenotypes of TT: deeply invading into the wall of renal vein/inferior vena cava (IVC) (DITT) or minimal/non-invasion (NITT), with different surgical strategy and post-thrombectomy outcome. However, the molecular characters of DITT/NITT have not been investigated, due to operational challenges and limited patient access. Methods: In this study, primary tumor and TT samples from 68 patients having IVC thrombectomy were prospectively collected for DNA mutation profiling and RNA expression profiling, using Predicine panel-based next generation sequencing and transcriptome RNA-SEQ, respectively. Next-generation sequencing and bioinformatics data analysis was conducted in Colleges of American Pathologist (CAP)-accredited laboratory in Shanghai, China (Huidu Shanghai Medical Sciences Ltd.). Kaplan–Meier survival analysis was performed to analyze the correlation between DITT/NITT and clinical outcome, and p-values were calculated using log-rank test. Results: When metastasis was detected, patients with DITT showed significantly shorter overall survival comparing to patients with NITT (P < 0.05, log-rank test). However, this difference was not observed in non-metastatic group. Successful sequencing has been achieved in 100% (68/68) patients. NITT tumor bulks more frequently carried mutations in PTEN, TP53 and epigenetic regulators such as SETD2, PBRM1 and KDM5C and had higher expression of cell cycle related pathway genes. In contrast, DITT had lower expression of genes encoding cell adhesion and extracellular matrix molecules such as ADAM33, NCAM1 and FLRT2, which could contribute to higher invasiveness. Conclusions: To our knowledge, this is the first large study to systematically interrogate genomic landscape and molecular features of different TT phenotypes in RCC patients. The differential genomic mutation and gene expression landscapes across the two groups may reflect biologic differences and clinical implications such as tumor invasiveness and aggressiveness.

ProP-PD for proteome-wide motif-mediated interaction discovery

Many protein–protein interactions are mediated by short linear motifs (SLiMs; 3–10 amino acid stretches; typically found in intrinsically disordered regions; low-to-mid micromolar affinities). The human proteome is expected to contain tens of thousands of SLiMs. However, to date only ~3000 SLiM instances have been discovered. The field required a scalable and accurate experimental approach to tackle SLiM discovery. Proteomic peptide-phage display (ProP-PD) is based on classical phage display updated with a combination of computational library design, custom oligonucleotide library synthesis, next-generation sequencing (NGS), and integrative data analysis.

Clinical and Molecular Characterization of POLE Mutations as Predictive Biomarkers of Response to Immune Checkpoint Inhibitors in Advanced Cancers.

PURPOSEDNA polymerase epsilon is critical to DNA proofreading and replication. Mutations in POLE have been associated with hypermutated tumors and antitumor response to immune checkpoint inhibitor (ICI) therapy. We present a clinicopathologic analysis of patients with advanced cancers harboring POLE mutations, the pattern of co-occurring mutations, and their response to ICI therapy within the context of mutation pathogenicity.METHODSWe conducted a retrospective analysis of next-generation sequencing data at MD Anderson Cancer Center to identify patient tumors with POLE mutations and their co-occurring mutations. The pathogenicity of each mutation was annotated using InterVar and ClinVar. Differences in therapeutic response to ICI, survival, and co-occurring mutations were reported by POLE pathogenicity status.RESULTSFour hundred fifty-eight patient tumors with POLE mutations were identified from 14,229 next-generation sequencing reports; 15.0% of POLE mutations were pathogenic, 15.9% benign, and 69.1% variant of unknown significance. Eighty-two patients received either programmed death 1 or programmed death ligand-1 inhibitors as monotherapy or in combination with cytotoxic T-cell lymphocyte-4 inhibitors. Patients with pathogenic POLE mutations had improved clinical benefit rate (82.4% v 30.0%; P = .013), median progression-free survival (15.1 v 2.2 months; P < .001), overall survival (29.5 v 6.8 months; P < .001), and longer treatment duration (median 15.5 v 2.5 months; P < .001) compared to those with benign variants. Progression-free survival and overall survival remained superior when adjusting for number of co-occurring mutations (≥ 10 v < 10) and/or microsatellite instability status (proficient mismatch repair v deficient mismatch repair). The number of comutations was not associated with response to ICI (clinical benefit v progressive disease: median 13 v 11 comutations; P = .18).CONCLUSIONPathogenic POLE mutations were associated with clinical benefit to ICI therapy. Further studies are warranted to validate POLE mutation as a predictive biomarker of ICI therapy.

NgsJulia: population genetic analysis of next-generation DNA sequencing data with Julia language

A sound analysis of DNA sequencing data is important to extract meaningful information and infer quantities of interest. Sequencing and mapping errors coupled with low and variable coverage hamper the identification of genotypes and variants and the estimation of population genetic parameters. Methods and implementations to estimate population genetic parameters from sequencing data available nowadays either are suitable for the analysis of genomes from model organisms only, require moderate sequencing coverage, or are not easily adaptable to specific applications. To address these issues, we introduce ngsJulia, a collection of templates and functions in Julia language to process short-read sequencing data for population genetic analysis. We further describe two implementations, ngsPool and ngsPloidy, for the analysis of pooled sequencing data and polyploid genomes, respectively. Through simulations, we illustrate the performance of estimating various population genetic parameters using these implementations, using both established and novel statistical methods. These results inform on optimal experimental design and demonstrate the applicabil- ity of methods in ngsJulia to estimate parameters of interest even from low coverage sequencing data. ngsJulia provide users with a flexible and efficient framework for ad hoc analysis of sequencing data.ngsJulia is available from: https://github.com/mfumagalli/ngsJulia

Identification of Survival-Specific Genes in Clear Cell Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

Purpose: Although mutations are associated with carcinogenesis, little is known about survival-specific genes in clear cell renal cell carcinoma (ccRCC). We developed a customized next-generation sequencing (NGS) gene panel with 156 genes. The purpose of this study was to investigate whether the survival-specific genes we found were present in Korean ccRCC patients, and their association with clinicopathological findings. Materials and Methods: DNA was extracted from the formalin-fixed, paraffin-embedded tissue of 22 ccRCC patients. NGS was performed using our survival-specific gene panel with an Illumina MiSeq. We analyzed NGS data and the correlations between mutations and clinicopathological findings and also compared them with data from the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) and Renal Cell Cancer-European Union (RECA-EU). Results: We found a total of 100 mutations in 37 of the 156 genes (23.7%) in 22 ccRCC patients. Of the 37 mutated genes, 11 were identified as clinicopathologically significant. Six were novel survival-specific genes (ADAMTS10, CARD6, NLRP2, OBSCN, SECISBP2L, and USP40), and five were top-ranked mutated genes (AKAP9, ARID1A, BAP1, KDM5C, and SETD2). Only CARD6 was validated as an overall survival-specific gene in this Korean study (p = 0.04, r = −0.441), TCGA-KIRC cohort (p = 0.0003), RECA-EU (p = 0.0005). The 10 remaining gene mutations were associated with clinicopathological findings; disease-free survival, mortality, nuclear grade, sarcomatoid component, N-stage, sex, and tumor size. Conclusions: We discovered 11 survival-specific genes in ccRCC using data from TCGA-KIRC, RECA-EU, and Korean patients. We are the first to find a correlation between CARD6 and overall survival in ccRCC. The 11 genes, including CARD6, NLRP2, OBSCN, and USP40, could be useful diagnostic, prognostic, and therapeutic markers in ccRCC.

A performance evaluation study: Variant annotation tools - the enigma of clinical next generation sequencing (NGS) based genetic testing

Dramatically expanding our ability for clinical genetic testing for inherited conditions and complex diseases such as cancer, next generation sequencing (NGS) technologies are allowing for rapid interrogation of thousands of genes and identification of millions of variants. Variant annotation, the process of assigning functional information to DNA variants based on the standardized Human Genome Variation Society (HGVS) nomenclature, is a fundamental challenge in the analysis of NGS data that has led to the development of many bioinformatic algorithms. In this study, we evaluated the performance of 3 variant annotation tools: Alamut® Batch, Ensembl Variant Effect Predictor (VEP), and ANNOVAR, benchmarked by a manually curated ground-truth set of 298 variants from the medical exome database at the Molecular Diagnostics Laboratory at Lurie Children's Hospital. Of the 3 tools, VEP produces the most accurate variant annotations (HGVS nomenclature for 297 of the 298 variants) due to usage of updated gene transcript versions within the algorithm. Alamut® Batch called 296 of the 298 variants correctly; strikingly, ANNOVAR exhibited the greatest number of discrepancies (20 of the 298 variants, 93.3% concordance with ground-truth set). Adoption of validated methods of variant annotation is critical in post-analytical phases of clinical testing.

Data Processing and Germline Variant Calling with the Sentieon Pipeline.

Public and private genomic sequencing initiatives generate ever-increasing amounts of genomic data creating a need for improved solutions for genomics data processing (Stephens et al.PLoS Biol 13:e1002195, 2015). The Sentieon® Genomics software enables rapid and accurate analysis of next-generation sequence data. In this work, we present a typical use of the Sentieon Genomics software for germline variant calling. The Sentieon germline variant calling pipeline produces more accurate results than other tools on third-party benchmarks (Katherine et al. Front Genet 10:736, 2019; Shen et al. bioRxiv, 885517, 2019) in one tenth the time of comparable pipelines. Parts of this guide come from the official Sentieon Genomics software manual in https://support.sentieon.com/manual (Sentieon. Sentieon Genomics software manual, n.d.) and from the official Sentieon Genomics software application notes in https://support.sentieon.com/appnotes (Sentieon. Sentieon Genomics software application notes, n.d.) and are republished with permission. For additional details and advanced usage instructions of the Sentieon tools, refer to the software manual.

Identification and expression analysis of ten novel small non-coding RNAs (sncRNAs) in cancer cells using a high-throughput sequencing approach.

Non-coding RNAs are characterized as RNA molecules, which lack the capacity to encode protein structures and appear to include a level of internal signals. Moreover, they control various stages of gene expression, thus controlling the cell physiology and development. In this study, we implemented a high-throughput sequencing approach based on the primary semi-conductor technology and computational tools, in order to identity novel small non-coding RNAs. Fourteen human cancer cell lines were cultured, and RNA samples were enriched for small RNAs following semi-conductor next generation sequencing (NGS). Bioinformatics analysis of NGS data revealed the existence of several classes of ncRNAs using the miRDeep* and CPSS 2.0 software. To investigate the existence of the predicted non-coding RNA sequences in cDNA pools of cell lines, a developed qPCR-based assay was implemented. The structure of each novel small ncRNA was visualized, using the RNAfold algorithm. Our results support the existence of twenty (20) putative new small ncRNAs, ten (10) of which have had their expression experimentally validated and presented differential profiles in cancerous and normal cells. A deeper comprehension of the ncRNAs interactive network and its role in cancer can therefore be translated into a wide range of clinical applications. Despite this progress, further scientific research from different perspectives and in different fields is needed, so that the riddle of the human transcriptome can be solved.

Abstract LBA005: Detection of KRAS amplification on extrachromosomal DNA (ecDNA) upon acquired resistance to KRASG12C inhibitors

Abstract The recent discovery and clinical validation of KRAS inhibitors (KRASi) has ushered in a new therapeutic approach to directly address the previously undruggable mutant KRAS-driven cancers. Unfortunately, as with other oncogene-directed therapies, acquired resistance to KRASi has been observed that is partially attributed to secondary mutations in KRAS and/or mutations or fusions in other mitogen-activated protein kinase (MAPK) signaling genes. Emerging evidence suggests that amplification of oncogenes on acentric extrachromosomal DNA (ecDNA) might constitute an important mechanism of rapid resistance to receptor tyrosine kinase (RTK) and MAPK targeting therapies. Therefore, we investigated whether resistance to KRASi monotherapy is mediated through an ecDNA mechanism. We previously noted focal gene amplification of KRAS and MET in recurrent tumors isolated from patients treated with the selective KRASG12C inhibitor, adagrasib. Analysis of next-generation sequencing (NGS) data from a patient tumor revealed evidence of circular DNA structures encompassing the KRAS locus. Consistent with this finding, interphase FISH analysis indicated the presence of KRAS amplifications on ecDNA in this adagrasib-treated patient tumor. To further interrogate this biology, we examined a genetically modified CT26 murine tumor model harboring KRASG12C alteration. Analogous to the clinical observations, both adagrasib as well as the recently approved KRASi sotorasib, induced transient tumor regressions that subsequently resumed growth following several weeks of continuous therapy. Strikingly, whole-genome sequencing data from isolated recurrent tumors revealed focal amplification of KRASG12C and metaphase FISH confirmed high levels of KRASG12C on ecDNA as compared to vehicle-treated samples. Further mechanistic studies are underway. Collectively, these observations implicate ecDNA as an important mediator of resistance to KRASi monotherapy and reinforce the critical need for novel therapeutic strategies to address ecDNA oncogene amplification-driven cancers. Citation Format: Ryan J Hansen, Steven Horton, Julie Wiese, BellJohn Bibay, E.Lorena Mora-Blanco, Nam-Phuong Nguyen, Lars D Engstrom, David M Briere, Andres Calinisan, Peter Olson, Mark M Awad, Andrew J Aguirre, Jason Christiansen, Christian Hassig, Shailaja Kasibhatla. Detection of KRAS amplification on extrachromosomal DNA (ecDNA) upon acquired resistance to KRASG12C inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA005.

Bacterial Community and Diversity from the Watermelon Cultivated Soils through Next Generation Sequencing Approach.

Knowledge and better understanding of functions of the microbial community are pivotal for crop management. This study was conducted to study bacterial structures including Acidovorax species community structures and diversity from the watermelon cultivated soils in different regions of South Korea. In this study, soil samples were collected from watermelon cultivation areas from various places of South Korea and microbiome analysis was performed to analyze bacterial communities including Acidovorax species community. Next generation sequencing (NGS) was performed by extracting genomic DNA from 92 soil samples from 8 different provinces using a fast genomic DNA extraction kit. NGS data analysis results revealed that, total, 39,367 operational taxonomic unit (OTU), were obtained. NGS data results revealed that, most dominant phylum in all the soil samples was Proteobacteria (37.3%). In addition, most abundant genus was Acidobacterium (1.8%) in all the samples. In order to analyze species diversity among the collected soil samples, OTUs, community diversity, and Shannon index were measured. Shannon (9.297) and inverse Simpson (0.996) were found to have the highest diversity scores in the greenhouse soil sample of Gyeonggi-do province (GG4). Results from NGS sequencing suggest that, most of the soil samples consists of similar trend of bacterial community and diversity. Environmental factors play a key role in shaping the bacterial community and diversity. In order to address this statement, further correlation analysis between soil physical and chemical parameters with dominant bacterial community will be carried out to observe their interactions.

Evaluation of copy number variants for genetic hearing loss: a review of current approaches and recent findings.

Structural variation includes a change in copy number, orientation, or location of a part of the genome. Copy number variants (CNVs) are a common cause of genetic hearing loss, comprising nearly 20% of diagnosed cases. While large deletions involving the gene STRC are the most common pathogenic CNVs, a significant proportion of known hearing loss genes also contain pathogenic CNVs. In this review, we provide an overview of currently used methods for detection of CNVs in genes known to cause hearing loss including molecular techniques such as multiplex ligation probe amplification (MLPA) and digital droplet polymerase chain reaction (ddPCR), array-CGH and single-nucleotide polymorphism (SNP) arrays, as well as techniques for detection of CNVs using next-generation sequencing data analysis including targeted gene panel, exome, and genome sequencing data. In addition, in this review, we compile published data on pathogenic hearing loss CNVs to provide an up-to-date overview. We show that CNVs have been identified in 29 different non-syndromic hearing loss genes. An understanding of the contribution of CNVs to genetic hearing loss is critical to the current diagnosis of hearing loss and is crucial for future gene therapies. Thus, evaluation for CNVs is required in any modern pipeline for genetic diagnosis of hearing loss.

次世代シーケンサーデータの解析手法 第 16 回 なぜ次から次へと新規手法が開発されるのか？

次世代シーケンサーデータの解析手法 第 16 回 なぜ次から次へと新規手法が開発されるのか？

次世代シーケンサーデータの解析手法 第 17 回 バイオインフォマティクス教育の今後

次世代シーケンサーデータの解析手法 第 17 回 バイオインフォマティクス教育の今後

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

Evaluation of the Oncomine Comprehensive Assay v3 panel for the detection of 1p/19q codeletion in oligodendroglial tumours

AimsAccurate assessment of 1p/19q codeletion status in diffuse gliomas is of paramount importance for diagnostic, prognostic and predictive purposes. While targeted next generation sequencing (NGS) has been widely implemented for...

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench.

Next-generation sequencing (NGS) has actualized the human papillomavirus (HPV) virome profiling for in-depth investigation of viral evolution and pathogenesis. However, viral computational analysis remains a bottleneck due to semantic discrepancies between computational tools and curated reference genomes. To address this, we developed and tested automated workflows for HPV taxonomic profiling and visualization using a customized papillomavirus database in the CLC Microbial Genomics Module. HPV genomes from Papilloma Virus Episteme were customized and incorporated into CLC “ready-to-use” workflows for stepwise data processing to include: (1) Taxonomic Analysis, (2) Estimate Alpha/Beta Diversities, and (3) Map Reads to Reference. Low-grade (n = 95) and high-grade (n = 60) Pap smears were tested with ensuing collective runtimes: Taxonomic Analysis (36 min); Alpha/Beta Diversities (5 s); Map Reads (45 min). Tabular output conversion to visualizations entailed 1–2 keystrokes. Biodiversity analysis between low- (LSIL) and high-grade squamous intraepithelial lesions (HSIL) revealed loss of species richness and gain of dominance by HPV-16 in HSIL. Integrating clinically relevant, taxonomized HPV reference genomes within automated workflows proved to be an ultra-fast method of virome profiling. The entire process named “HPV DeepSeq” provides a simple, accurate and practical means of NGS data analysis for a broad range of applications in viral research.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (&amp;gt;84%) of concordance rate for ploidy and sex, sensitivity (&amp;gt;88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (&amp;gt;5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable