Whole Genome Amplification Products Research Articles

Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer

Abstract Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character. Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs. Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts. Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression. Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6561.

Simultaneous Isolation and Amplification of mRNA and Genomic DNA of a Single Cell.

Many biological or pathological processes are driven by cells difficult to identify or isolate, i.e., rare cells. Very often, these cells have elusive biology. Therefore, their detailed characterization is of utmost importance. There are many approaches that allow analysis of few or even many targets within one class of biomacromolecules/analytes (e.g., DNA, RNA, proteins, etc.) in single cells. However, due to rarity of the cells of interest, there is a great need to comprehensively analyze multiple analytes within these cells, in other words to perform multi-omics analysis. In this chapter, I describe a method to isolate, separate, and amplify total mRNA and genomic DNA of a single cells, using whole transcriptome (WTA) and whole genome amplification (WGA). These WTA and WGA products enable simultaneous analysis of transcriptome and genome of a single cell using various downstream high-throughput approaches.

Exploration of a Novel Noninvasive Prenatal Testing Approach for Monogenic Disorders Based on Fetal Nucleated Red Blood Cells.

Due to technical issues related to cell-specific capture methods, amplification, and sequencing, noninvasive prenatal testing (NIPT) based on fetal nucleated red blood cells (fNRBCs) has rarely been used for the detection of monogenic disorders. Maternal peripheral blood was collected from 11 families with hereditary hearing loss. After density gradient centrifugation and cellular immunostaining for multiple biomarkers, candidate individual fetal cells were harvested by micromanipulation and amplified by whole-genome amplification (WGA). Whole-exome sequencing/whole-genome sequencing (WGS) and Sanger sequencing were performed on the identified fNRBCs to determine the fetal genotype. The impact of single-cell and pooled WGA products on the sequencing quality and results was compared. A combined analysis strategy, encompassing whole-exome sequencing/WGS, haplotype analysis, and Sanger sequencing, was used to enhance the NIPT results. fNRBCs were harvested and identified in 81.8% (9/11) of families. The results of cell-based-NIPT (cb-NIPT) were consistent with those of invasive prenatal diagnosis in 8 families; the coincidence rate was 88.9% (8/9). The combined analysis strategy improved the success of cb-NIPT. The overall performance of pooled WGA products was better than that of individual cells. Due to a lack of alternative fetal cells or sufficient sequencing data, cb-NIPT failed in 3 families. We developed a novel fNRBC-based NIPT method for monogenic disorders. By combining multiple analysis strategies and multiple fetal cell WGA products, the problem of insufficient genome information in a single cell was remedied. Our method has promising prospects in the field of NIPT for the detection of monogenic disorders.

Application of conventional IVF during preimplantation genetic testing for aneuploidies: a feasibility study

Research questionIs it feasible to apply conventional IVF to couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) with non-severe male infertility? DesignThe last wash fluid of biopsied trophectoderm (TE) cells was collected for whole genome amplification (WGA). A method was developed to determine parental contamination. Using single-nucleotide polymorphism (SNP) analysis, two standard curves were established; further mixtures were used for verification. Finally, 29 WGA products from couples undergoing conventional IVF were used to evaluate parental contamination. ResultsThe WGA results of the last wash fluid of biopsied TE cells revealed almost no free DNA. By adopting two strategies based on maternally and paternally biased SNP in the mixture, data from bioinformatics analysis were analysed to determine the relationship between maternal (Index M) and paternal (Index F) bias statistics. Two standard curves were successfully established based on these indices that allowed the prediction of maternal and parental contamination, which correlated well with actual ratios of known composition mixtures during validation. The average contamination level was 10.6% determined from 10 WGA products that featured maternal contamination, whereas that of the other 19 products that featured paternal contamination was less than 10%. ConclusionsThis study confirmed the feasibility of applying conventional IVF to couples undergoing PGT-A with non-severe male infertility.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/μL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

Single-cell genotyping of phytoplankton from ocean water by gel-based cell manipulation.

A comprehensive understanding of phytoplankton diversity is valuable for assessing an environment of interest as phytoplankton are primary producers in various aquatic food webs. Microscopic analyses are useful for diversity assessment based on characteristic cell morphologies. However, phylogenetic classification based solely on morphology requires an extremely high level of expertise. The genetic approach is another option for evaluating phytoplankton diversity; however, it cannot reveal morphological information. To integrate these two approaches, an original technology was developed, that is referred to as microcavity array (MCA)/gel-based cell manipulation (GCM). The model experiments using monocultures of various phytoplankton indicated that the efficiencies of cell recovery and isolation of single-cell plankton were dependent on cell size and shape. Cells with widths larger than the cavity width showed high level of recovery and isolation efficiency. Subsequent whole-genome amplification (WGA) of isolated single-cell plankton provided a sufficient amount (≈30μg) of WGA products for genetic analyses. Furthermore, it is showed that MCA/GCM could directly analyze phytoplankton in ocean water obtained from Suruga Bay, Japan, without any cumbersome pretreatment. These results indicate that MCA/GCM technology is a powerful tool for elucidating the phytoplankton diversity in marine environment.

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic‐activated cell sorting system, and CD45‐negative and cytokeratin‐positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole‐genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4‐ALK fusion (17/20 cells, 85%) with an alectinib‐resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA‐associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA‐associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.

Genetic, Prenatal, and Preimplantation Genetic Diagnosis of Glutaric Aciduria Type I

Purpose: Glutaric Acid Type I (GA-I) is an inherited metabolic disorder. Although the treatment guidelines for GA-I were established a decade ago, they cannot block the vertical heredity. We aim to apply genetic methods to block the inheritance of GA-I and verifies the efficiency of Next-Generation Sequencing (NGS)-based Preimplantation Genetic Testing for Monogenic disease (PGT-M) of GA-I.Materials and methods: A non-consanguineous Chinese family was diagnosed with GA-I by Sanger sequencing. PGT-M and prenatal diagnosis (PND) were performed for the carrier. 5 blastocysts were used for the trophectoderm biopsy. After Whole-Genome Amplification (WGA), the WGA products were used for Sanger sequencing, NGS-based PGT-M and PGT-A. Sanger sequencing-based PND was performed in second trimester to confirm the results of PGT-M.Results: A compound heterozygous mutation was diagnosed in the GCDH gene with co-segregation. One is [c.533G>A (p.G178E)] and another is [c.914C>T (p.S305L)]. 2 blastocysts were diagnosed as normal and one of them was transferred into the mother’s uterus. Finally, a healthy female was born 39 weeks after transplantation.Conclusion: Our study successfully applied NGS-based PGT-M to avoid GA-I and highlights the efficiency of genetic diagnoses. It has significant implications on genetic counseling and genetic diagnosis for GA-I.

Development of High Throughput Single-Cell Analysis System for Circulating Tumor Cells Based on Digital Micromirror Device

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst.

BackgroundA key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm. The envelope proteins of HERV-W and -FRD (human endogenous retrovirus-W and -FRD), syncytin-1 and syncytin-2, are mainly distributed in the placenta, and play important roles in the development of the placenta. The placenta originates from the trophectoderm of the blastocyst. It is unclear whether the envelope proteins of HERV-W and -FRD have an effect on the development of the trophectoderm and whether they have any association with the implantation of the blastocyst.MethodsThe whole-genome amplification products of the human blastocyst trophectoderm were used to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, and included age, body mass index (BMI), basic follicle stimulating hormone(bFSH), rate of primary infertility and oligo-astheno-teratospermia, the thickness of the endometrium on the day of endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR.ResultsThe relative copy number of syncytin-1 in the pregnant group (median: 424%, quartile: 232%–463%, p < 0.05) was significantly higher than in the non-pregnant group (median: 100%, quartile: 81%–163%). There was a correlation (rs = 0.681, p < 0.001) between the copy number of syncytin-1 and blastocyst implantation after embryo transfer. As the stem cells differentiated, the expression of NANOG mRNA decreased, and the expression of caudal type homeobox 2(CDX2) and β-human chorionic gonadotropin (β-hCG) mRNAs increased. Compared to the undifferentiated cells, the relative expression of the syncytin-1 mRNA was 1.63 (quartile: 0.59–6.37, p > 0.05), 3.36 (quartile: 0.85–14.80, p > 0.05), 10.85 (quartile: 3.39–24.46, p < 0.05) and 67.81 (quartile: 54.07–85.48, p < 0.05) on day 1, 3, 5 and 7, respectively, after the differentiation. The relative expression of syncytin-2 was 5.34 (quartile: 4.50–10.30), 7.90 (quartile: 2.46–14.01), 57.44 (quartile: 38.35–103.87) and 344.76 (quartile: 267.72–440.10) on day 1, 3, 5 and 7, respectively, after the differentiation (p < 0.05). The copy number of syncytin-1 increased significantly during differentiation.ConclusionPreceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.

Abstract 2702: Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells

Abstract Background: Clonal evolution and heterogeneity are among the factors that make treatment of Multiple Myeloma (MM) challenging, and disease monitoring requires invasive bone marrow biopsies. Tumor heterogeneity has previously been demonstrated at copy-number level, showing patterns of recurring Copy-Number Alterations (CNA). Conversely, Loss-of-Heterozygosity (LoH) profiling has been investigated less extensively. Presence of genomic patterns of LoH has been associated with Homologous Recombination Deficiency (HRD) in MM, suggesting a role for therapy with PARP inhibitors. Here we report the genome-wide LoH profiling in single-CMMCs isolated from enriched peripheral blood of four different MM patients using a non-invasive approach that combines CellSearch® and DEPArray™ technology. Methods: CMMCs were obtained from peripheral blood of four MM patients using CellSearch for enrichment and DEPArray NxT for isolation. CMMCs enrichment was obtained using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. After single cell isolation, Ampli1™ Whole Genome Amplification (WGA) kit was used to amplify single-cell genomic DNA. Whole genome sequencing libraries were prepared from WGA products using Ampli1™ LowPass kit and low-pass sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBiosuite. Single-cell genome-wide LoH profiles were obtained by a custom algorithm based on the ratio between mono- and bi-allelic loci in non-overlapping segments of uniform copy-number. Results: Out of 315 cells analyzed from the four MM patients, 244 (77%) showed LoH events. Extensive LoH regions were detected in all patients, with 231 cells showing copy-neutral LoH events and 121 showing LoH events in copy-gain regions. Globally, 460 copy-neutral LoHs, and 292 LoHs in copy-gain regions were detected. Two highly conserved copy-neutral events (9p; 16q) were detected in one of the patients analyzed, suggesting they are early events. Interestingly, the first of the 2 events includes CDKN2A, a tumor suppressor which has been implicated in predisposition to MM. Notably, in a second patient, chromosome 11 is associated both to a copy-neutral LoH and to an LoH event in a copy-gain segment including the CCND1 gene, whose amplification in MM has been associated with multidrug resistance expression. Conclusions: Here we present a non-invasive workflow for the molecular characterization of single CMMCs isolated from peripheral blood. Specifically, we reported the presence of chromosomal or sub-chromosomal LoH events in all MM patients, suggesting a pervasive phenomenon in MM which deserves to be further explored as potential therapy target. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Paola Tononi, Marianna Garonzi, Valentina del Monaco, Andrea Raspadori, Steven Gross, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2702.

A comparative study of whole genome amplification and low-template DNA profiling

Generation of high-quality STR profiles from trace amounts of cells remains challenging. Although increasing the number of PCR cycles improves the sensitivity of STR analysis, stochastic effects may complicate interpretation and deem the profiles inconclusive. Whole Genome Amplification (WGA) has been successfully employed to improve the outcome of subsequent STR analysis by increasing the number of available DNA template. However, there is limited information on how WGA performs in comparison to the elevated PCR cycles.This study aims to evaluate the quality of STR profiles obtained from 10 and 25 pg of DNA that had undergone WGA, against that from 32-PCR cycle protocol. Buccal cell DNA was subjected to 16-h WGA using REPLI-g® Single Cell Kit followed by 28-cycle STR amplification with AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit. The results showed that the WGA yielded significantly lower allele detection rates than the 32-cycle PCRs, although the mean peak height ratios were comparable. Few allelic drop-ins were also observed in profiles derived from the WGA products, while none was present in the 32-cycle PCR profiles. These preliminary data suggested that the WGA protocol examined did not offer any improvement on DNA profile quality over the elevated PCR cycle method. However, the success rates of the WGA in this study seemed to contradict the previous reports, thus modifications to the protocol used would be further investigated.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Abstract 439: Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells

Abstract Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel. Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite. Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was &amp;lt; 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis. Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.

Limited sensitivity of NGS-based detection of unbalanced products from reciprocal translocations inpolar bodies and blastocysts

Introduction Numerous publications have demonstrated that preimplantation genetic diagnosis (PGD) increases the implantation rate and percentage of healthy newborns or at least decreases themiscarriage rate for translocation carriers with recurrent pregnancy losses (Fisher et al. 2010, Ikumaet al. 2018). The aim of this study was to evaluate whether VeriSeq PGS, a next generation sequencingbasedmethod for aneuploidy screening, is also suitable for the detection of structural aberrations inpolar bodies and trophectoderm cells. Since the product provider didn´t validate the method forunbalanced structural changes, our goal was to determine its sensitivity. Material & methods Array-based preimplantation genetic diagnostic was performed on polar bodies obtained from 12 women as well as on trophectoderm samples from five couples being translocation carriers. Furthermore, polar bodies from four women carrying other structural aberrations (i.e.versions, insertions, duplications) were included in the study. Whole genome amplification (WGA)was performed using the SurePlex DNA Amplification System. Cy3-labelled sample DNA and Cy5-labelled male reference DNA were hybridized for 4-16 hours on 24sure+ arrays. The slides were washedand scanned according to the manufacturer´s protocol. NGS libraries (VeriSeq PGS, Illumina Inc.) wereprepared from the same WGA products and sequenced on a MiSeq as described by the manufacturer. Data obtained with both methods were analyzed using the BlueFuse Multi Software. Fisher's exact testwas applied for statistical analyses. Results Reciprocal translocations present on a total of 18 chromosomes were included in this study. The results obtained with next generation sequencing in general delivered an excellent signal-to- noiseratio and a high dynamic range. Although, no statistically significant differences in the detection ofstructural variants between aCGH and NGS were observed, the comparison of both methods revealedseveral discordant results. In three cases, only one out of two translocated segments was detectableby NGS, whereas the aCGH method identified both fragments. By contrast, in the cohort with otherstructural aberrations, a 17 Mb insertion of chromosome 3 was detected only by NGS. Conclusions Our data indicate that although it is possible to analyze structural aberrations in singlecells with VeriSeq PGS, this method has some limitations in the detection of small telomericimbalances. The detection of structural aberrations is not only determined by their size but moreimportantly, it is locus-dependent. Hence, unbalanced reciprocal translocations with both translocatedfragments smaller than 8 Mb containing repetitive or paralogous sequences, might not be detectablewith VeriSeq PGS resulting in false negative results. Therefore, PGD for such translocations requires aprevalidation by e.g. sequencing of single sperm cells or unbalanced products of conception in orderto determine the locus-specific detection sensitivity.

The comparison of two whole genome amplification methods: A new method of pre-implantation genetic testing (PGT-M) for Southeast Asian deletion in alpha-thalassemia using high resolution melting analysis

Introduction In Southeast Asia, alpha-thalassemia with Southeast Asian (SEA) deletion type is the most common mutations of alpha-thalassemia-1 which can cause severe form known as Bart's hydrops fetalis. For couples which carrier of single gene defects, pre-implantation genetic testing for monogenic (PGT-M) plays a role in genetic testing for selecting of unaffected embryos prior implantation. Recently, high resolution melting (HRM) analysis is a simple, rapid and widely used for genotyping and gene scanning of genetic variation. In this study, we developed HRM analysis method for detection of SEA deletion in alpha-thalassemia and compared two WGA methods, SurePlex DNA Amplification System and REPLI-g Single Cell on the performance of HRM analysis and linkage analysis. Material and Methods Retrospective study was carried out in a single private fertility clinic from 2015 to 2017. The single cell embryos were biopsied on blastocyst stage from couples with disease- carrier of SEA deletion type. The trophectoderm biopsied cells were amplified by two whole genome amplification (WGA), SurePlex DNA Amplification System-PCR based method (Illumina, USA) and REPLI-g MDA Single Cell (QIAGEN, Germany). All WGA products were analyzed with two primer pairs of HRM analysis for detection of SEA deletion type in alpha-thalassemia disease and confirmed with the informative short tandem repeated (STRs) linkage analysis. Results A total of 53 single cell biopsied were amplified with SurePlex-WGA (n=33) and REPLI-g MDA (n=20) which used as template for HRM analysis. The HRM results revealed clearly distinguished between wild type and mutant type of SEA deletion mutation in alpha-thalassemia. Comparing between two WGA methods, the HRM amplification results efficiency and allele drop out (ADO) rate of REPLI- g MDA were 95% (19/20) and 31.57% (6/19), respectively. Meanwhile, for SurePlex-WGA showed 75.76% (25/33) and 28% (7/25), respectively. Furthermore, the HRM melting profile results of REPLI-g MDA showed higher concordance rate with the informative STRs linkage analysis results (63.16%, 12/18) than that of SurePlex-WGA (48%, 12/25). Conclusions HRM analysis is an alternative technique with high sensitivity and accuracy for detecting of alpha-thalassemia mutations. The REPLI-g MDA Single Cell method with HRM analysis is suitable for detection of alpha-thalassemia1 SEA type deletion in PGT-M and provided a good concordance with informative STRs linkage analysis. However, ADO from PCR amplification could be occurred and caused of misdiagnosis which might be affected to the interpretation results of the embryos disease- status. Thus, the mutation analysis method using HRM analysis combined with STRs linkage analysis are recommend in PGT-M to avoid the effected of ADO and increase the accuracy and reliable of the PGT-M results.

Pregnancy following exclusion testing for SCA3 on polar bodies in a fresh cycle: a comparison of STR-based targeted haplotyping and genome-wide SNP analysis and meiomapping

Introduction Preimplantation genetic testing of monogenic disease (PGT-M) is allowed in Germany only with approval from the federal ethics committee and autosomal dominant disorders are often rejected. Polar body analysis, however, does not require ethical approval. A 34-year-old asymptomatic patient with a family history of spinocerebellar ataxia type 3 (SCA3) applied for PGT-M on trophectoderm samples but was rejected. The patient's father has a repeat expansion in the ATXN3 gene and is affected with SCA3, a progressive autosomal dominantly inherited neurological disease characterized by ataxia, spasticity and ocular movement abnormalities. As the patient does not want to know her status, we offered exclusion testing based on polar body analysis in a fresh cycle and have compared targeted haplotyping by STR analysis with genome-wide SNP genotyping and meiomap analysis. Material and methods The patient and her father were genotyped for 12 STR markers within 0.44 Mb of the ATXN3 gene on 14q32. 11 STRs were heterozygous (9 fully informative) and used for phasing high and low risk haplotypes. Eggs were collected (Day 0) and the first polar body (PB1) biopsied from mature MII oocytes. ICSI was then performed and PB2 biopsied from three fertilised zygotes on Day 1. After whole genome amplification (WGA) (Repli-g Single Cell Kit; Qiagen, Venlo, Netherlands) multiplex PCR was performed and the products run on a capillary sequencer. Haplotypes were constructed based on the pre-case workup. In addition, genome-wide SNP genotyping of the DNAs from the patient, her father and the WGA products from the polar bodies was carried out (HumanKaryomap-12 SNP array; Illumina, San Diego, USA) for maternal haplotyping and meiomap analysis. Results WGA and multiplex PCR was successful for all of the polar bodies except for the PB2 of Oocyte 1. As all 11 closely linked informative STRs were homozygous for the high-risk haplotype in PB1 from this oocyte, the corresponding PB2 and oocyte was deduced to have the low risk haplotype assuming normal segregation during meiosis. For Oocytes 2 and 3, the PB1 was heterozygous for most STRs and the PB2 of these oocytes were homozygous for the high and low risk haplotypes, respectively. Therefore, the embryos derived from Oocytes 1 and 2 were deduced to have the low risk maternal haplotype while the embryo from Oocyte 3 should have the high-risk haplotype. On Day 5, the blastocysts arising from Oocytes 1 and 2 were transferred, resulting in an ongoing pregnancy (currently week 10). Genome-wide SNP genotyping and meiomap analysis was concordant with STR analysis in all cases. Conclusions We report a pregnancy following targeted STR-based haplotyping and exclusion testing of an autosomal dominant disease by polar body analysis in a fresh cycle. Genome-wide SNP genotyping and meiomap analysis was fully concordant with the STR results but had the advantage that it also demonstrated that segregation of the maternal chromosome 14 was normal and predicted that the embryos from the two oocytes in which both polar bodies were analysed were euploid for all maternal chromosomes.

DOPlify, Target Sequence Enrichment and Allele Drop-Out – is there a benefit?

Introduction The ability to combine whole genome amplification (WGA) with gene-specific primers in a single PCR reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) is an advantage of DOPlify® and the RHS targeted sequence enrichment (TSE) protocol. This study aims to compare Allele Drop-Out (ADO) rates by comparing three different approaches; gene specific PCR only, WGA and WGA with TSE. Material and Methods Thirty 5-cells aliquots were manually sorted from a cell line containing a 2bp heterozygous BRCA1 mutation (GM14090, Coriell Institute). To allow an assessment of aneuploidy detection, thirty 5-cell aliquots from an aneuploid cell line (GM04965, 48,XXY,+21, Coriell Institute) were also used. In the TSE approach, cells were amplified using DOPlify® (RHS Ltd) with the inclusion of three BRCA1 primers. One BRCA1 primer set was designed to specifically amplify the region on chromosome 17 containing the deletion site in GM14090 and other two primer sets to amplify BRCA1 linker markers. The resulting WGA-TSE product was diluted 1/10 and used as template for a second gene specific multiplex PCR (Qiagen) containing the same BRCA1 primers. The BRCA1 PCR products were then pooled back into the WGA-TSE product before library preparation and analysed using a single library index (WGA-TSE+GS). Secondly, for PGT-M only, cells were subjected to two rounds of gene specific PCR using multiplex PCR (Qiagen) and the same BRCA1 primers (GS+GS). Lastly, cells were amplified according to standard DOPlify® protocol (RHS Ltd) with no BRCA1 primers added. The resulting WGA product was diluted 1/10 and seeded into the BRCA1 multiplex PCR reaction (Qiagen) (WGA+GS). PCR products from the WGA-TSE+GS condition were sequenced according to standard PG-Seq™ protocol (RHS Ltd). Amplicons from the GS+GS and WGA+GS conditions were diluted 1/10 before final library pooling then were sequenced with the Illumina MiSeq. Results The 2bp heterozygous BRCA1 deletion was accurately detected in 10/10 of the WGA-TSE+GS samples, 10/10 of the GS+GS samples and 7/10 of the WGA+GS samples with average allele frequencies of 41%, 47% and 53%, respectively. The average read depth across the 2bp deletion site was 120, 129 and 138, respectively. One GM04965 sample from the GS+GS condition failed to amplify the region. Correct aneuploidy results were obtained for 9/9 BRCA1 cells and 10/10 GM04965 cells processed with the WGA-TSE+GS protocol with one BRCA1 sample excluded after failing quality control (weak amplification). The GS+GS protocol does not allow for PGT-A assessment. Conclusions The DOPlify® TSE protocol is able to reliably generate both PGT-A and PGT-M results from a cell sample representing a single embryo biopsy. The PGT-A results were not compromised, and PGT-M results showed no ADO and were comparable to the gene specific PCR protocol. These results highlight the benefit of the DOPlify® TSE protocol, which is now being clinically validated.

Simultaneous detection of chromosomal aneuploidy and a monogenic disease by next-generation sequencing with linkage analysis

Introduction Mutations giving rise to single gene disorders (SGD) are currently directly detected in PGD-M using PCR and Sanger sequencing. However, amplification failures, allelic drop-out (ADO), mosaicism and contamination limits PGT precision and diagnostic efficiency. The current approaches for PGT-M generally uses standard panels pre-designed for each disease. The present study applied whole genome amplification (WGA) of biopsied trophectoderm cells and concurrent next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping on an Ion Torrent Personal Genome Machine (PGM) with the aim of customized testing for each family. Material and Methods Exome sequencing data of a consanguineous family applying for ART (mother, father and child), who give their informed consent, was used to establish the haplotype of the thalassemia gene locus-based on informative SNPs by using the VcfTools and R software. A total of 88 loci containing 116 SNPs located 2 Mb upstream and 2 Mb downstream of the beta globin gene locus (HBB) were selected for NGS-based SNP haplotyping. These loci were then submitted to a primer design website ( www.ampliseq.com ; Thermo Fisher Scientific). The WGA products of three embryos generated during the IVF cycle of the couple and which were diagnosed as aneuploid and genomic DNA of the child were used for the Amplicon Library Preparation (using Ion AmpliSeq™ Library kits) for PGT-M and in parallel for PGT-A. To validate the SNP-based haplotype, a STR markers’-based haplotype was also established. Results The SNP-based haplotypes of the tested embryos and of the child were successfully constructed by NGS with linkage analysis, using GeneHunter v2.15 software. Out of the three embryos tested with family customized PGT-M panel, the first embryo was found to be haploidentical with the child for the HBB locus. The second embryo shared only the paternal allele, whereas the third embryo was carrying the other parental alleles when compared to the child's alleles. These haplotypes were also verified by classical STR marker-based haplotyping. Read depth of amplicons ranged between 70 and 6800. The parallel PGS diagnoses of the three embryos were identical with the original diagnoses of the respective embryos. The variations of SNP call rates and read depths of the trophectoderm samples were correlated with library concentrations. Conclusions In conclusion, this approach allows for a successful simultaneous detection of chromosomal aneuploidy and a monogenic disease by NGS with linkage analysis. Especially in but not limited to consanguineous families, establishing family-specific gene panels for PGT-M maximizes allelic information with a minimum number of SNPs without the need for direct mutation testing thus lowering the cost of testing per embryo.

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform

BackgroundRecent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S.MethodsThe artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH).ResultsOur SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable.ConclusionsOur studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.