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Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-021-00889-9.

STUDY QUESTION What is the true incidence of chromosomal mosaicism in embryos analyzed by preimplantation genetic testing (PGT) SUMMARY ANSWER The true incidence of chromosomal mosaicism is much lower than we usually surmise. WHAT IS KNOWN ALREADY In recent years, contemporary methods for chromosome analysis, along with the biopsy of more than one cell, have given rise to an increased rate of chromosomal mosaicism detection after preimplantation genetic testing for aneuploidy. However, the exorbitant incidence of mosaicism represents a dilemma and imposes restrictions on the application of PGT treatment. Concern has been raised about the possibility that the incidence of chromosomal mosaicism is overestimated and quite a few of the results are false-positive errors. However, studies verifying the diagnosis of chromosomal mosaicism and assessing the true incidence of chromosomal mosaicism are limited. STUDY DESIGN, SIZE, DURATION A total of 1719 blastocysts from 380 patients who underwent PGT treatment were retrospectively analyzed to evaluate the typical incidence of mosaicism. Then 101 embryos donated by 70 couples were re-biopsied and dissected into three portions if available: trophectoderm (TE), inner cell mass (ICM), and the remaining portions. All the portions were tested using next-generation sequencing (NGS), and the results were compared to the original diagnosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The setting for this study was a university-affiliated center with an in-house PGT laboratory. All samples were amplified with multiple annealing and looping-based amplification cycles (MALBACs) and the NGS was carried out on a Life Technologies Ion Proton platform. MAIN RESULTS AND THE ROLE OF CHANCE A clinical TE biopsy revealed an incidence of 11.9% for diploid-aneuploid mosaicism (DAM), 17.3% for aneuploid mosaicism (AM) and 29.1% in total. After rebiopsy, 94.1% whole-chromosome aneuploidies and 82.8% segmental-chromosome aneuploidies were confirmed in the embryos. As for the mosaic errors, only 32 (31.7%) out of 101 embryos presented with uniform chromosomal aberrations in agreement with the original biopsy results, 15 (14.8%) embryos presented with de novo chromosomal aberrations, and 54 (53.5%) embryos showed a euploid profile in all portions. Among the 32 uniform embryos, the true mosaicism was confirmed in only 4 cases, where a reciprocal chromosomal aberration was identified; 14 embryos presented with identical mosaicism, providing the moderate evidence for true mosaicism; and 14 embryos displayed uniform full aneuploidies in all portions of embryo, revealing a high-grade mosaicism or a false-negative diagnosis. Logistical regression analysis revealed that the concordance rate with ICM was associated with the type and level of mosaicism. The concordance rate of segmental-chromosome mosaicism was significantly lower than whole-chromosome mosaicism (adjusted Odds Ratio (aOR): 5.137 (1.061, 24.876), P = 0.042) and compared to DAM, the concordance rate of AM was significantly higher (aOR: 6.546 (1.354, 31.655), P = 0.019). The concordance rate also increased with increasing levels of mosaicism (P LIMITATIONS, REASONS FOR CAUTION This study was limited by a small sample size and the use of a single whole-genome amplification (WGA) method and NGS platform. These findings are only applicable to samples subjected to MALBAC amplification and Ion Proton platform, and studies involving larger sample sizes and multiple WGA methods and NGS platforms are required to prove our findings. WIDER IMPLICATIONS OF THE FINDINGS TE biopsy is reliable to detect whole-chromosome aneuploidies, but the ability to diagnose mosaicism is doubtful. More attention should be paid to false-positive and false-negative errors in NGS-based PGT, especially for laboratories using less stringent criteria for mosaicism classification (i.e. 20–80%), which might be subject to a much higher false-positive mosaicism rate in the practice. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the National Key R&D Program of China (No. 2016YFC1000206-5) and the National Natural Science Foundation of China (No. 81701509) TRIAL REGISTRATION NUMBER N/A.

The genome‑wide copy number analysis of circulating tumor cells (CTCs) provides a promising prognostic biomarker for survival in breast cancer liver metastasis (BCLM) patients. The present study aimed to confirm the prognostic value of the presence of CTCs in BCLM patients. We previously developed an assay for the genome‑wide pattern differences in copy number variations (CNVs) as an adjunct test for the routine imaging and histopathologic diagnosis methods to distinguish newly diagnosed liver metastases and recurrent liver metastases. Forty‑three breast cancer patients were selected for this study in which 23 newly diagnosed and 20 recurrent liver metastases were diagnosed by histopathology and 18F‑FDG PET/CT imaging. CTCs were counted from all patients using the CellSearch system and were confirmed by cytomorphology and three‑color immunocytochemistry. Genomic DNA of single CTCs was amplified using multiple annealing and looping based amplification cycles (MALBAC). Then, we compared the CTC numbers of newly diagnosed and recurrent BCLM patients using Illumina platforms. A high CTC frequency (>15 CTCs/7.5 ml blood) was found to be correlated with disease severity and metastatic progression, which suggests the value for CTCs in the diagnosis of BCLM in comparison with pathohistology and PET/CT imaging (P>0.05). Moreover, CTCs isolated from BCLM patients remained an independent prognostic detection factor associated with overall survival (P=0.0041). Comparison between newly diagnosed and recurrent liver metastases revealed different frequencies of CNVs (P>0.05). Notably, the CNV pattern of isolated CTCs of recurrent BCLM patients was similar to recurrent liver metastases (nearly 82% of the gain/loss regions). Functional enrichment analysis identified 25 genes as a CNV signature of BCLM. Among them, were defensin and β‑defensin genes, which are significantly associated with anti‑angiogenesis and immunomodulation signaling pathways. High CTC frequencies are effective in the evaluation and differentiation between newly diagnosed liver metastases from recurrent liver metastases. Future clinical studies will be necessary to fully determine the prognostic potential of CTC cluster signatures in patients with BCLM.

PURPOSE: To explore the whole-chromosome status, origins, and mechanisms of chromosomal abnormalities in good-quality cleavage embryos using multiple annealing and looping-based amplification cycle (MALBAC) sequencing. METHODS: The embryos studied came from7 patients (maternal aged 26–35) who had healthy birth from the same IVF cycles. These 21 frozen day 3 good-quality embryos were thawed and disaggregated into individual blastomere. Each blastomere was collected and analyzed by MALBAC sequencing. RESULTS: Conclusive results were obtained from a high percentage of blastomeres (95.3%). A total of 46.6% of blastomeres were diploid, 53.4% were abnormal, and 28.0% had complex aneuploidy. Out of 21 embryos, 3 (14.3%) were normal and 18 (85.7%) were mosaics, showing the occurrence of mitotic errors; aneuploidy was confirmed in all cells of 4 of the 18 embryos, which showed the coexistence of meiotic errors. Conclusive results were obtained from all blastomeres of 15 embryos (71.4%, 15/21), which enabled us to reconstruct the cell lineage on the basis of the chromosomal content of the blastomeres in each division. There were 9 mitotic errors (8.7%, 9/103): nondisjunction accounted for 88.9% (8/9), and endoreplication accounted for 11.1% (1/9). CONCLUSIONS: In good-quality embryos, there was a high rate and diverse array of chromosomal abnormalities. Morphological evaluation does not appear to assist in the reduction in meiotic errors from parental origins. Mitotic errors were common, and nondisjunction was found to be the main mechanism causing malsegregation during the cleavage divisions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01803-9) contains supplementary material, which is available to authorized users.

PURPOSE: To perform a preliminary exploration of a new embryo rank in clinical practice by combining the embryo chromosome copy number and mitochondrial copy number analysis of DNA extracted from embryo culture medium and blastocoel fluid. METHOD: Eighty-three ICSI embryos from day 2 or day 3 were cultured to day 5 or day 6. Thirty-two blastocysts of 3 cc or above were obtained. Culture medium and blastocoel fluid were collected at 24 h before blastocyst formation. The genomic DNA and mitochondrial DNA (mtDNA) from the culture medium combined with blastocoel fluid and the whole blastocyst were amplified and sequenced by MALBAC-NGS. We compared the chromosomal information generated by the new protocol from the culture medium and the information employed by the whole embryo method. A multivariable linear regression was performed to study the impact of the blastocyst morphological score, chromosomal abnormality, embryo mtDNA copy number, and female age on the culture medium mtDNA copy number. RESULTS: (1) The DNA from 31 blastocysts was successfully amplified, and the successful amplification rate was 96.9% (31/32). The success rate of the amplification of genomic DNA extracted from the culture medium was 87.5% (28/32). (2) There were 18 blastocysts in which the less invasive method and the whole embryo method revealed the same results. The consistency rate was 66.7% (18/27). (3) The culture medium mitochondrial DNA copy number (MCN) had a significantly positive correlation with the blastocyst mitochondrial DNA copy number (P = 0.001), female age (P = 0.012), and blastocyst score (P = 0.014), but there was no obvious correlation with blastocyst chromosome (P = 0.138). CONCLUSIONS: The preliminary exploration result of the less invasive approach for having an embryo rank was not satisfying, which still awaits further long-term evaluation.

Acral melanoma (AM) is an extremely aggressive subtype of melanoma that is prevalent in eastern Asia. AM exhibits high intertumoral and intratumoral heterogeneities with poor prognosis. To associate the genomic heterogeneities with phenotypic traits and efficacy of treatments, a method is needed to recover genomic information from limited samples with high specificity and sensitivity from early stage AM specimens. We performed laser capture microdissection to isolate single micro-tumor nests, containing only dozens of cells, from stained tissue slices and then applied multiple annealing and looping-based amplification cycles, a highly efficient whole-genome amplification method originally developed for single cells, to amplify the whole genome of each tumor nest for sequencing. We were able to accurately profile the landscape of copy number alterations and single nucleotide variations of every single micro-tumor nest and to quantitatively characterize the heterogeneities at different levels, between tumor and nevi, among patients, among different phenotypes within a same tumor, and among adjacent tumor cell clusters with identical phenotypic appearance. We have found that genomic heterogeneity exists extensively and that branched evolution happens in the early stage of AM development. We are able to build the phylogenetic tree among these phenotypically addressable cell clusters.

Methylmalonic acidemia combined with homocysteinemia and cobalamin C type (MMA-CblC, MIM # 277400) is a rare inherited disease with cobalamin metabolic disorder, which are caused by deficiency in the MMACHC gene. A couple with a proband child carried with compound heterozygous mutations of MMACHC (c.609G>A and c.567 dup T, NM_015506) sought for assisted reproductive technology to avoid the transmission of pathogenic genetic variants and unnecessary induction of labor. Thus, in vitro fertilization (IVF), preimplantation genetic testing (PGT), and prenatal genetic diagnosis were applied to fulfill this clinical demand. In this study, seven embryos were biopsied and carried out whole-genome amplification using multiple annealing and looping-based amplification cycle (MALBAC) method. Sanger sequencing together with copy number variation (CNV) analysis and single-nucleotide polymorphism (SNP) haplotyping was conducted to detect the mutated alleles and chromosomal abnormalities simultaneously. Three embryos (E07, E06, and E02) were confirmed without CNVs and inherited mutations at MMACHC gene. Embryo E07 with the best embryo ranking of 5BB was selected preferentially to transfer which led to a successful pregnancy and an unaffected live birth. Prenatal genetic diagnosing with amniotic fluid cells, Sanger sequencing with cord blood cells, and neonate MMA screening further verified our successful application of PGT in preventing mutated allele transmission for this rare inherited disease.

Nowadays, most of the preimplantation genetic testing (PGT) is performed with a strategy of comprehensive chromosome screening and trophectoderm biopsy. Nevertheless, patients with ovarian insufficiency may not have competent blastocysts. In the present study, we aimed to establish the value of multiple annealing and looping-based amplification cycle (MALBAC)-based next-generation sequencing (NGS) for PGT in day-3 embryos. A total of 94.3% (1168/1239) of embryos yielded informative results, and the overall embryo euploid rate was 21.9% (256/1168). Overall, 225 embryos were transferred in 169 cycles with a clinical pregnancy rate of 49.1% (83/169). The live birth and implantation rates were 47.3% (80/169) and 44.4% (100/225), respectively. Double embryos transfer showed higher clinical pregnancy and live birth rates compared with single embryo transfer, but the implantation rates were similar (44.2% vs. 44.6%,P> 0.05). The euploid rate for reciprocal translocations (16.1%) was significantly lower than that for Robertsonian translocations (28.0%,P< 0.01) and inversions (28.0%,P< 0.01). However, higher percentages of embryos withde novoabnormalities were observed with Robertsonian translocations (23.3%,P< 0.01) and inversions (30.5%,P< 0.01) than with reciprocal translocations (11.6%). We demonstrated that NGS for PGT on day-3 embryos is an effective clinical application, particularly for patients with a diminished ovarian reserve and limited embryos.

BackgroundIdentification of de novo mutations from cell populations requires single-cell whole-genome sequencing (SCWGS). Although many experimental protocols of SCWGS have been developed, few computational tools are available for downstream analysis of different types of somatic mutations, including copy number variation (CNV).ResultsWe developed SCCNV, a software tool for detecting CNVs from whole genome-amplified single cells. SCCNV is a read-depth based approach with adjustment for the whole-genome amplification bias.ConclusionsWe demonstrate its performance by analyzing data collected from most of the single-cell amplification methods, including DOP-PCR, MDA, MALBAC and LIANTI. SCCNV is freely available at https://github.com/biosinodx/SCCNV.

The aim of this study is to explore the relationship between mitochondrial DNA (mtDNA) copy number and embryo implantation potential in in vitro fertilization (IVF). A retrospective study of 319 blastocysts from patients undergoing preimplantation genetic testing (PGT) at Reproductive Medicine Center in Tongji Hospital from January 2016 to February 2018 was conducted. We used multiple annealing- and looping-based amplification cycles (MALBAC) technology to amplify the genetic materials from the trophectoderm cells of blastocysts, and next-generation sequencing (NGS) technology to test mitochondrial DNA copy number. Box-Cox transformation was introduced to eliminate the skewness distribution of mtDNA copy number, and the transformed data were defined as adjusted mtDNA. Subsequently, associations between adjusted mtDNA and the clinical characteristics of patients were assessed by univariate analysis and multiple linear regression. In addition, Gaussian Naive Bayes classifier was also used to predict pregnancy outcomes. We observed that only antral follicle count (AFC) was significantly associated with adjusted mtDNA without the influence of multicollinearity. What's more, the distribution of the adjusted mtDNA of blastocysts resulting in live birth was more concentrated than that of others. The area under the curve (AUC) of the prediction model that combined adjusted mtDNA with other clinical characteristics of patients was up to 0.81, higher than that excluded adjusted mtDNA. Among patient clinical characteristics, AFC was significantly associated with adjusted mtDNA. Mitochondrial DNA copy number may help to optimize the pregnancy outcome prediction in IVF.

Background To assess whether preimplantation genetic testing for aneuploidy with next generation sequencing (NGS) outweighs single nucleotide polymorphism (SNP) array in improving clinical outcomes. Methods A retrospective analysis of the clinical outcomes of patients who underwent PGT-A treatment in a single center from January 2013 to December 2017.A total of 1418 couples who underwent PGT-A treatment were enrolled, of which 805 couples used NGS for PGT-A, while the remaining 613 couples used SNP array for PGT-A. Clinical pregnancy rate, miscarriage rate and healthy baby rate were compared between the MALBAC-NGS-PGT-A and MDA-SNP-PGT-A groups. Results After testing karyotypes of 5771 biopsied blastocysts, 32.2% (1861/5771) were identified as chromosomally normal, while 67.8% were chromosomally abnormal. In terms of clinical outcomes, women in the MALBAC-NGS-PGT-A group had a significantly higher clinical pregnancy rate (50.5% vs 41.7%, p = 0.002) and healthy baby rate (39.6% vs 31.4%, p = 0.003), and a lower miscarriage rate (15.5% vs 22.8%, p = 0.036). Conclusion This is the largest study reporting the extensive application of NGS-based PGT-A, whilst comparing the clinical outcomes of MALBAC-NGS-PGT-A and MDA-SNP-PGT-A. The results provide greater evidence supporting the wider use of NGS in PGT-A, not only for its lower cost but also for its improved clinical outcomes compared to SNP-based PGT-A.

Whole genome amplification (WGA) is crucial for whole genome sequencing to investigate complex genomic alteration at the single-cell or even single-molecule level. Multiple displacement amplification (MDA) and multiple annealing and looping based amplification cycles (MALBAC) are two most widely applied WGA methods, which have different advantages and disadvantages, dependent on research objectives. Herein, we compared the MDA and MALBAC to provide more information on their performance in droplets and tubes. We observed that the droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than the conventional tube method. Furthermore, the droplet method exhibited higher efficiency and sensitivity for both homozygous and heterozygous single nucleotide variants (SNVs) at the low sequencing depth. In addition, we also found that MALBAC offered a greater uniformity and reproducibility and MDA showed a better efficiency of genomic coverage and SNV detection. Our results provided insights that will allow future decision making.

Mitochondria host multiple copies of their own small circular genome that has been extensively studied to trace the evolution of the modern eukaryotic cell and discover important mutations linked to inherited diseases. Whole genome and exome sequencing have enabled the study of mtDNA in a large number of samples and experimental conditions at single nucleotide resolution, allowing the deciphering of the relationship between inherited mutations and phenotypes and the identification of acquired mtDNA mutations in classical mitochondrial diseases as well as in chronic disorders, ageing and cancer. By applying an ad hoc computational pipeline based on our MToolBox software, we reconstructed mtDNA genomes in single cells using whole genome and exome sequencing data obtained by different amplification methodologies (eWGA, DOP-PCR, MALBAC, MDA) as well as data from single cell Assay for Transposase Accessible Chromatin with high-throughput sequencing (scATAC-seq) in which mtDNA sequences are expected as a byproduct of the technology. We show that assembled mtDNAs, with the exception of those reconstructed by MALBAC and DOP-PCR methods, are quite uniform and suitable for genomic investigations, enabling the study of various biological processes related to cellular heterogeneity such as tumor evolution, neural somatic mosaicism and embryonic development.

PURPOSE: To study the chromosome constitution of equal-sized three-cell embryo. METHODS: We determined the chromosome constitution of 105 blastomeres from 35 embryos using multiple annealing and looping-based amplification cycles (MALBAC) together with NGS sequencing technology. Chromosomal copy number variation (CNV) analysis was successfully performed in 27 embryos. We also analyzed radius, perimeter, area, and volume of each blastomere to explore the possibility of selecting the normal embryos. RESULTS: Majority of the embryos (77.8%, 21/27) studied were mosaic or aneuploid, and only 22.2% (6/27) had normal chromosome numbers. The aneuploid chromosomes spread across all chromosomes and the most frequent aneuploidies were for chromosomes 1, 16, and 18 followed by 13, 19, and 21. Statistical analyses showed no significant difference between euploid and aneuploid embryos regarding radius, perimeter, area, and volume of their blastomeres. CONCLUSIONS: Our results showed that majority of the equal-sized three-cell embryos were chromosomally abnormal and could not be distinguished by morphology observation, so they should be given lower priority at selection for transfer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1362-7) contains supplementary material, which is available to authorized users.

Circulating tumour cell (CTC) behaviours are distinct from those of bulk tissues. Thus, treatments to eliminate CTCs differ from the regimens followed to reduce the primary tumour and its metastases. Accordingly, comprehensively deciphering the single nucleotide variant (SNV) profiles in CTCs, which partially determine CTC behaviours, is a priority. Using viable CTCs isolated with the oHSV1-hTERT-GFP virus coupled with fluorescence-activated cell sorting (FACS), the whole genome was amplified using the multiple annealing and looping-based amplification cycle (MALBAC) method. CTC behaviours were evaluated using the SNVs found to be recurrently mutated in different cells (termed CTC-shared SNVs). Analysis of the sequencing data of 11 CTCs from 8 patients demonstrated that SNVs accumulated sporadically among CTCs and their matched primary tumours (22 co-occurring mutated genes were identified in the exomes of CTCs and their matched primary tissues and metastases), and 394 SNVs were shared by at least two CTCs. Mutated APC and LRP1B genes co-occurred in CTC-shared and bulk-tissue SNVs. Additionally, the breast-originating identity of the CTC-shared SNVs was verified, and they demonstrated the following CTC behaviours: i) intravasation competency; ii) increased migration or motility; iii) enhanced cell-cell interactions; iv) variation in energy metabolism; v) an activated platelet or coagulation system; and vi) dysfunctional mitosis. These results demonstrated that it is feasible to capture and amplify the genomes of single CTCs using the described pipeline. CTC-shared SNVs are a potential signature for identifying the origin of the primary tumour in a liquid biopsy. Furthermore, CTCs demonstrated some behaviours that are unique from those of bulk tissues. Therefore, therapies to eradicate these precursors of metastasis may differ from the existing traditional regimens.

To draw robust conclusions when trace DNA samples are detected in complex cases, it is essential to successfully recover and genotype short tandem repeats (STRs) from trace DNA. However, obtaining complete STR profiles by the conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) method is generally difficult as trace DNA is often less than 100 pg. Previous studies have proven that through whole-genome amplification (WGA), the yield of DNA from trace DNA samples could be improved. In this study, we used two WGA kits, namely, REPLI-g® Single Cell kit and MALBAC® Single Cell DNA Quick-Amp Kit (hereafter referred to as REPLI and MALBAC), to amplify DNA samples with a series of dilutions (from 5.00 ng/μL to 0.391 pg/μL). Typing of STR markers in samples with and without WGA were then performed on a CE platform by the application of Goldeneye® DNA ID System 20 A kit, as well as directly calling sequences from massive parallel sequencing (MPS) for WGA samples with 1.00 ng, 125 pg and 25.0 pg as DNA inputs. Quantification results demonstrated that the yield of samples with WGA could reach the microgram level. The amplification fold was at least > 2000 and > 200 for REPLI and MALBAC, respectively. CE results showed that the number of correctly called loci was improved for trace DNA after WGA when the DNA inputs were lower than 25.0 pg for REPLI and 6.25 pg for MALBAC, respectively. WGA remarkably improved the percentage of called loci with DNA inputs lower than 50.0 pg, although poor performance in repeatability was observed. MPS results suggested that the correctly called loci calculated by MPS reads were mostly more than those calculated by CE, particularly for those of short length, implying MPS of samples after WGA is worth testing in the future. In conclusion, WGA has the potential usability for forensic trace DNA analysis at the single-cell level with good fidelity, although its repeatability requires further improvement.

Autosomal dominant polycystic kidney disease (ADPKD, autosomal dominant PKD or adult-onset PKD) is the most prevalent and potentially lethal kidney disease that is hereditary and lacks effective treatment. Preimplantation genetic diagnosis (PGD) of embryos in assistant reproductive technology (ART) helps to select mutation-free embryos for blocking ADPKD inheritance from the parents to their offspring. However, there are multiple pseudogenes in the PKD1 coding region, which make blocking ADPKD inheritance by PGD complicated and difficult. Therefore, this technique has not been recommended and used routinely to ADPKD family plan. Here, we report a new strategy of performing PGD in screening (target-) mutation-free embryos. We firstly used a long-range PCR amplification and next generation sequencing to identify the potential PKD1 mutant(s). After pathogenic variants were detected, multiple annealing and looping-based amplification cycles (MALBAC), a recently developed whole genome amplification method, was used to screen embryo cells. We successfully distinguished the mutated allele among pseudogenes and obtained mutation-free embryos for implantation. The first embryo transfer attempt resulted in a healthy live birth free of ADPKD condition and chromosomal anomalies which was confirmed by aminocentesis at week 18 of gestation, and by performing live birth genetic screening. The first MALBAC-PGD attempt in ADPKD patient resulted in a healthy live birth free of ADPKD and chromosomal anomalies. MALBAC-PGD also enables selecting embryos without aneuploidy together and target gene mutation, thereby increasing implantation and live birth rates.

Robertsonian translocation (RT) is a common cause for male infertility, recurrent pregnancy loss, and birth defects. Studying meiotic recombination in RT-carrier patients helps decipher the mechanism and improve the clinical management of infertility and birth defects caused by RT. Here we present a new method to study spermatogenesis on a single-gamete basis from two RT carriers. By using a combined single-cell whole-genome amplification and sequencing protocol, we comprehensively profiled the chromosomal copy number of 88 single sperms from two RT-carrier patients. With the profiled information, chromosomal aberrations were identified on a whole-genome, per-sperm basis. We found that the previously reported interchromosomal effect might not exist with RT carriers. It is suggested that single-cell genome sequencing enables comprehensive chromosomal aneuploidy screening and provides a powerful tool for studying gamete generation from patients carrying chromosomal diseases.

Objective To assess the relationship between human blastocyst chromosomal ploidy established by niPGT-A and increasing age. Methods This is a prospective multicenter study carried out by ten assisted reproduction centers after their embryologists acquired training and validated their results with the previous use of niPGT-A. A total of 94 couples with indication for niPGT-A due to increase maternal age, male factor, repeated implantation failures, recurrent abortion or because they requested niPGT-A were included in this study. The couples had no karyotype abnormalities. After ICSI, the embryos were cultured until blastocyst stage using one or two step culture systems, single or sequential media respectively, at 37°C in an atmosphere of 6-7% CO2 and 5-20% O2 incubators. On day 3, we re-evaluated cleavage embryos to complete cumulus cells removal. The embryos were then cultured in individual well, with 20µl of medium under oil until they reached blastocyst stage. The blastocysts were vitrified and stored in liquid nitrogen. After that, the spent blastocyst culture medium (20µl) was transferred to a PCR tube and sent for analysis in the genetic laboratory, where it was stored at -80°C until sequencing. A total of 243 samples of spent blastocyst culture medium were collected on the 5th/6th day. Cell-free DNA secreted on culture medium was amplified using NICS Sample Preparation Kit (Yikon Genomics), based on the MALBAC technology. After whole genome amplification, the DNA was measured using a Qubit 2.0 fluorometer and subjected to next generation sequencing (NGS) using Illumina MiSeq® platform. The data were analyzed using the ChromGo® software (Yikon Genomics). Results The mean age of the patients was 38±4.08 years with an interval of 20-44 years. The euploid was diagnosed in 36.4% (80/220) of cases, aneuploidy in 31.3% (69/220), and mosaicism in 32.3% (71/220; with ≥60% aneuploidy) of blastocysts. Mosaic values ranged from 29.8% to 33.8% in different age groups. Individually, the most frequent chromosomal abnormality was XXY (Klinefelter Syndrome) occurring in 18 cases, followed by chromosome 21 (trisomy/monosomy) in 8 cases. The niPGT-A data showed a ≥60% incidence of aneuploid cells in all cases of chromosomal mosaicism (n=71). Conclusion A high degree of mosaicism with aneuploidy cells was detected, and some hypotheses were suggested for this data (niPGT-A sensitivity in detecting the self-correction of chromosomal abnormalities phenomenon). However, it did not vary remarkably with age. On the other hand, euploidy levels had a negative correlation with age and aneuploidy levels had a positive relationship. This is the first report in the literature to relate chromosomal ploidy in blastocysts using niPGT-A and increasing patient age.

Single-cell DNA sequencing is a powerful tool to evaluate the state of heterogeneity of heterogeneous tissues like cancer in a quantitative manner that bulk sequencing can never achieve. DOP-PCR (Degenerate Oligonucleotide-Primed Polymerase Chain Reaction), MDA (Multiple Displacement Amplification), MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), LIANTI (Linear Amplification via Transposon Insertion) and TnBC (Transposon Barcoded) have been the primary choices to prepare single-cell libraries. TnBC library prep method is a simple and versatile methodology, to detect copy number variations or to obtain the absolute copy numbers of genes per cell.

// Jiawei Xu 1, * , Wenbin Niu 1, * , Zhaofeng Peng 1 , Xiao Bao 1 , Meixiang Zhang 1 , Linlin Wang 1 , Linqing Du 1 , Nan Zhang 1 , Yingpu Sun 1 1 The First Affiliated Hospital of Zhengzhou University, Centre for Reproductive Medicine, Zhengzhou, Henan 450000, China * These authors have contributed equally to this work Correspondence to: Yingpu Sun, email: syp2008@vip.sina.com Keywords: triploidy, SNP array, next generation sequencing, MALBAC, preimplantation genetic diagnosis and screen Received: September 23, 2016 Accepted: October 14, 2016 Published: November 09, 2016 ABSTRACT Triploidy occurred about 2-3% in human pregnancies and contributed to approximately 15% of chromosomally caused human early miscarriage. It is essential for preimplantation genetic diagnosis and screen to distinct triploidy sensitively. Here, we performed comparative investigations between MALBAC-NGS and MDA-SNP array sensitivity on triploidy detection. Self-correction and reference-correction algorism were used to analyze the NGS data. We identified 5 triploid embryos in 1198 embryos of 218 PGD and PGS cycles using MDA-SNP array, the rate of tripoidy was 4.17‰ in PGS and PGD patients. Our results indicated that the MDA-SNP array was sensitive to digyny and diandry triploidy, MALBAC-NGS combined with self and reference genome correction strategies analyze were not sensitive to detect triploidy. Our study demonstrated that triploidy occurred at 4.17‰ in PGD and PGS, MDA-SNP array could successfully identify triploidy in PGD and PGS and genomic DNA. MALBAC-NGS combined with self and reference genome correction strategies were not sensitive to triploidy.

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Objective To validate a 24-chromosome aneuploidy preimplantation genetic screening protocol based on multiple annealing and looping-based amplification cycle (MALBAC) and next-generation sequencing (NGS). Design Single-nucleotide polymorphism (SNP) array and MALBAC-NGS analysis. Setting University-affiliated in vitro fertilization (IVF) center. Patient(s) Fifteen women from whom 30 blastocysts were obtained for genotyping. Intervention(s) Not applicable. Main Outcome Measure(s) Chromosomal status comparison of results of array comparative genomic hybridization (aCGH), SNP array, and MALBAC-NGS for 24-chromosome aneuploidy screening. Result(s) Trophectoderm biopsy samples from blastocysts were first analyzed using array comparative genomic hybridization (aCGH); the embryos with detected with chromosomal abnormalities were rebiopsied, and dissociated into two portions, and subjected to SNP array and MALBAC-NGS for 24-chromosome aneuploidy screening. All 30 samples were successfully genotyped by array CGH, SNP array, and MALBAC-NGS. All blastocysts were correctly identified as aneuploid, and there was a 100% concordance in terms of diagnosis provided between the three methods. In the 720 detected chromosomes, the concordance rate between MALBAC-NGS and array CGH was 99.31% (715 of 720), and the concordance rate between MALBAC-NGS and SNP array was 99.58% (717 of 720). When compared with aCGH, MALBAC-NGS specificity for aneuploidy call was 99.85% (674 of 675; 95% CI, 99.17–99.97) with a sensitivity of 91.11% (41 of 45; 95% CI, 79.27–96.49). When compared with SNP array, MALBAC-NGS specificity for aneuploidy call was 99.85% (676 of 677; 95% CI, 99.17–99.97) with a sensitivity of 95.35% (41 of 43; 95% CI, 85.54–98.72). Conclusion(s) MALBAC-NGS provides concordant chromosomal results when compared with aCGH and SNP array in blastocysts with chromosomal abnormalities.

The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although GenomePlex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.

Whole genome amplification (WGA) facilitates the metagenomics study of low biomass environments, with the risks of introducing biases and community skewing. The objective of this study was therefore to compare two common WGA methods in terms of community distortion. Multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) were evaluated with three contrasting soils and by 16S rRNA genes amplicon and whole genome shotgun sequencing. Both WGA technologies yielded stable amplification from nanogram quantities of soil DNA. The MiSeq sequencing of 16S rRNA genes demonstrated considerable loss of species richness and highly skewed community profiles for MDA-derived templates compared to the actual communities. Bacillales of low GC genome were consistently enriched in all MDA libraries. In contrast, MALBAC captured the community differences between the three soils as revealed by a hierarchical clustering analysis, and the community distortion was moderate in terms of bacterial richness. Unlike MDA, MALBAC may enrich bacteria of high GC genomes. Prokaryotic community analysis of the shotgun data confirmed the findings obtained by the 16S rRNA amplicon sequencing. Despite the wide use of MDA, these findings implicate MALBAC as promising in characterizing microbial communities in low biomass soils and highlights the potential of community profile as a quality control for WGA of environmental samples.

STUDY QUESTION Is minimally invasive chromosome screening (MICS) using blastocyst culture medium (BCM) sufficiently fast and accurate for preimplantation genetic testing (PGT) SUMMARY ANSWER A new assay for MICS, named MICS-Inst achieved high-resolution, comprehensive chromosome ploidy detection using BCM. WHAT IS KNOWN ALREADY BCM is a viable source of genomic DNA for use in PGT. STUDY DESIGN, SIZE, DURATION Forty-one vitrified blastocysts donated by 22 couples known to carry a chromosome rearrangement and 21 vitrified blastocysts donated from 8 couples with normal karyotypes were used in this study. Good-quality blastocysts, defined as Day 5 and Day 6 embryos ≥ BB (AA, AB, BA, BB) based on the Gardner system were used for analysis. Recruitment took place from May 2018 to August 2018. We performed PGT for structural rearrangements (PGT-SR) on 41 BCM, trophectoderm (TE) biopsy and blastocyst-stage embryo (BE) samples as well as PGT for aneuploidies (PGT-A) on 21 BCM, TE biopsy and BE samples. PARTICIPANTS/MATERIALS, SETTING, METHODS We made several significant modifications to the BCM composition (mixing blastocoel fluid and spent blastocyst medium) as well as the pre-existing multiple annealing and looping-based amplification cycles (MALBAC) techniques and library generation procedures. The design of a quasilinear preamplification (Pre-AMP) primer and AMP primers 1 and 2 enables the preparation of a next-generation sequencing library after the exponential amplification stage by introducing the Illumina P5 and P7 primers into the final products, which are then ready for sequencing. Sequencing was performed on the Illumina Hiseq 2500 platform with 2.0 Mb raw reads generated for each sample. MAIN RESULTS AND THE ROLE OF CHANCE For PGT-A, BCM and TE biopsy samples showed 90% and 86% clinical concordance with the corresponding BE samples, respectively. In addition, both BCM and TE biopsy samples showed 76% karyotype concordance with the corresponding BE samples. For PGT-SR, we successfully obtained ploidy information for all 23 chromosomes with the exception of any rearrangements involving the Y chromosome. Both BCM and TE biopsy samples showed 100% clinical concordance with the corresponding BE samples in detecting chromosomal rearrangements. BCM and TE biopsy samples showed 90% and 100% karyotype concordance with the corresponding BE samples, respectively. Additionally, no statistically significant differences were detected in the aforementioned values of the BCM and TE biopsy samples in either PGT-A or PGT-SR (P > 0.05). Moreover, we achieved accurate quantification of segmental abnormalities using BCM samples. In addition, MICS-Inst reduced the number of steps required for library preparation through the use of new primer designs, resulting in an overall time reduction of 7.5 h. This time reduction allows for the performance of fresh blastocyst transfers. LIMITATIONS, REASONS FOR CAUTION The main limitation is that BE, rather the inner cell mass, was used as the standard to evaluate the chromosome screening results. WIDER IMPLICATIONS OF THE FINDINGS These results show that MICS-Inst is effective in procedure and precision for PGT, and that it is possible to achieve fresh blastocyst transfer following PGT. The implications are significant, as these findings may lead to minimally invasive PGT methods in the future. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (No. 81671423 and No. 81402130), the National Key Research and Development Program of China (No. 2018YFC1003100), Liaoning Provincial Key Research and Development Program (No. 2018225090), the Fok Ying Tung Education Foundation (No. 151039) and Distinguished Talent Program of Shengjing Hospital (No. ME76). No competing interests declared.

Single-cell genomics is an alluring area that holds the potential to change the way we understand cell populations. Due to the small amount of DNA within a single cell, whole-genome amplification becomes a mandatory step in many single-cell applications. Unfortunately, single-cell whole-genome amplification (scWGA) strategies suffer from several technical biases that complicate the posterior interpretation of the data. Here we compared the performance of six different scWGA methods (GenomiPhi, REPLIg, TruePrime, Ampli1, MALBAC, and PicoPLEX) after amplifying and low-pass sequencing the complete genome of 230 healthy/tumoral human cells. Overall, REPLIg outperformed competing methods regarding DNA yield, amplicon size, amplification breadth, amplification uniformity –being the only method with a random amplification bias–, and false single-nucleotide variant calls. On the other hand, non-MDA methods, and in particular Ampli1, showed less allelic imbalance and ADO, more reliable copy-number profiles and less chimeric amplicons. While no single scWGA method showed optimal performance for every aspect, they clearly have distinct advantages. Our results provide a convenient guide for selecting a scWGA method depending on the question of interest while revealing relevant weaknesses that should be considered during the analysis and interpretation of single-cell sequencing data.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

DNA typing using trace DNA poses a significant challenge for forensic science. To test whether whole genome amplification (WGA) technology could resolve this problem, we first evaluated the efficiency of WGA technology for DNA using the MALBAC® Single-cell DNA Kit to amplify trace DNA and the Goldeneye™ DNA ID System to genotype short tandem repeats (STRs). The amplified DNA yield and the STR-typing success rate were then assessed. The DNA yield was found to increase more than 200-fold following WGA. Moreover, the STR-typing success rate using the amplified DNA with a template

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

In this study, we aimed to assess the performance of two whole-genome amplification methods, multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycle (MALBAC), for β-thalassemia genotyping and single-nucleotide polymorphism (SNP)/copy-number variant (CNV) detection using two DNA sequencing assays. We collected peripheral blood, cell lines, and discarded embryos, and carried out MALBAC and MDA on single-cell and five-cell samples. We detected and statistically analyzed differences in the amplification efficiency, positive predictive value, sensitivity, allele dropout (ADO) rate, SNPs, and CV values between the two methods. Through Sanger sequencing at the single-cell and five-cell levels, we showed that both the amplification rate and ADO rate of MDA were better than those using MALBAC, and the sensitivity and positive predictive value obtained from MDA were higher than those from MALBAC for β-thalassemia genotyping. Using next-generation sequencing (NGS) at the single-cell level, we confirmed that MDA has better properties than MALBAC for SNP detection. However, MALBAC was more stable and homogeneous than MDA using low-depth NGS at the single-cell level for CNV detection. We conclude that MALBAC is the better option for CNV detection, while MDA is better suited for SNV detection.

Single cell whole genome sequencing helps to decipher the genome heterogeneity within a cell population and facilitates the analysis of trace amounts of genetic material, such as is found in human embryos. The mitochondrial genome, although an important part of the genetic composition of eukaryotic cells, is often neglected in single cell genome analysis. A recently developed single cell whole genome amplification method was used, known as multiple annealing and looping based amplification cycles (MALBAC-NGS), for simultaneous analysis of chromosomal and mitochondrial genomes at the single cell level. The platform was validated by a series of technical and biological replicates and used for chromosomal and mitochondrial copy number analysis in 399 in-vitro fertilized embryos from 81 couples. A positive correlation of maternal age with increased mitochondria quantity (β = 0.176, P = 0.001) was observed after adjusting for the impact of cell type. Lower numbers of mitochondria were detected in successfully implanted embryos, although the difference was not significant. It is proposed that MALBAC-NGS could potentially be used for an advanced pre-implantation genetic screening procedure with both chromosomal constitution and mitochondrial copy number being evaluated.

With the development and clinical application of genomics, more and more concern is focused on single-cell sequencing. In the process of single-cell sequencing, whole genome amplification is a key step to enrich sample DNA. Previous studies have compared the performance of different whole genome amplification (WGA) strategies on Illumina sequencing platforms, but there is no related research aimed at Ion Proton platform, which is also a popular next-generation sequencing platform. Here by amplifying cells from six cell lines with different karyotypes, we estimated the data features of four common commercial WGA kits (PicoPLEX WGA Kit, GenomePlex Single Cell Whole Genome Amplification Kit, MALBAC Single Cell Whole Genome Amplification Kit, and REPLI-g Single Cell Kit), including median absolute pairwise difference, uniformity, reproducibility, and fidelity, and examined their performance of copy number variation detection. The results showed that both MALBAC and PicoPLEX could yield high-quality data and had high reproducibility and fidelity; and as for uniformity, PicoPLEX was slightly superior to MALBAC.

Introduction For investigating the detection success rate and accuracy of non-invasive capacity screening(NICS) and determining its effectiveness on improvement of embryo transfer, we reviewed the research results and clinical advances of MALBAC-based NICS from multiple groups worldwide between 2016 to 2018. Material and Methods 1. Detection success rate: qPCR was used to investigate the starting DNA amount limit and the detection success rate of NICS in 1483 culture medium samples. 2. Detection accuracy 246 embryos with morphological scoring over CC were collected. Culture medium samples were subjected to NICS and the results were compared with whole embryo's aneuploidy results to evaluate the accuracy of NICS as an option of pre-implantation chromosome screening. Also, NICS were applied to 41 families with chromosome translocations to determine its accuracy on pre-implantation chromosome diagnosis. 3. Clinical validation MCT: 120 patients were enrolled and RCT were implemented for non-inferiority comparison of clinical outcomes between NICS and PGS. And 72 patients were recruited for superiority study by comparing clinical outcomes between traditional morphology assessment and NICS. Results 1. The overall success rate reached to 94.6% among all 1483 culture medium samples with NICS and it mounted to 99.2% when DNA amount ≥ 3pg. 2. The NPV of NICS were over 90% during pre-implantation chromosome screening and this value reached to 97% when applying NICS to pre-implantation chromosome diagnosis. The other performances of NICS including sensitivity, specificity, PPV were comparable to PGS. 3. The clinical results of NICS and PGS from 120 patients did not exhibit significant differences. The clinical outcomes of 72 cases showed that the miscarriage rate of NICS is 0%, and the results are still updating. Conclusions NICS is a novel genetic testing that allows an accurate selection of blastocysts with better developmental potential for transfer and might improve the clinical outcomes.

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing DNA amplification and library preparation methods are expensive and introduce coverage bias during sample pre-amplification. Based on a massively parallel 5184-microwell chip, we developed a high-throughput unbiased single-cell DNA library preparation approach up to 1800 cells for copy number variation (CNV) detection, which provides a comprehensive, scalable solution for revealing genome heterogeneity. Compared with existing methods, it is more efficient and cost-effective to construct a barcoded-single-cell library without pre-amplification in 2 days. Besides, it has significantly lower Median Absolute Pairwise Difference (MAPD:0.17±0.004) values which are used to evaluate the amplification biases and noises, than using MDA (multiple displacement amplification) method (MAPD: 0.79±0.23) or MALBAC (multiple annealing and looping-based amplification cycles) approach (MAPD : 0.24±0.002). We further successfully applied this approach to obtain single-cell CNV information of more than 500 cells from a tumor sample of hepatocellular carcinoma in one run. Using the unsupervised clustering, we identified at least two clonal subpopulations with a multitude of non-integer alterations across the genome. Overall, this is an unbiased, effective, high-throughput and low cost approach to figure out tumor heterogeneity and understand clonal evolution in tumor at the single-cell level. Citation Format: Liang Wu, Yuzhou Wang, Miaomiao Jiang, Shiping Liu. High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma [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 3519.