Multiple Annealing And Looping-based Amplification Cycles Research Articles

Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia.

To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Article. fifty-two deletional α-thalassemia couples. Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia.

The Comparison of Two Whole-Genome Amplification Approaches for Noninvasive Preimplantation Genetic Testing (ni-PGT) and the Application Scenario of ni-PGT during the Fresh Cycle

Recently, noninvasive preimplantation genetic testing (ni-PGT) using degenerate oligonucleotide primer PCR (DOP-PCR) and multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) methods has demonstrated predictable results in embryo testing. However, a considerable heterogeneity of results has been reported in numerous studies on these two WGA methods. Our aim was to evaluate the current WGA method for ni-PGT while further clarifying the applicable scenarios of ni-PGT in the fresh cycle. A total of 173 embryos were tested with trophectoderm biopsy and ni-PGT. In the whole preimplantation genetic testing, the clinical concordance rates of the detection results of DOP-PCR and MALBAC with the corresponding trophectoderm biopsy results were 64.12% (84/131) and 68.99% (89/129), respectively (P=0.405). However, in the detection of abnormal embryos, the detection efficiency of ni-PGT is significantly improved [MALBAC: 96.55% versus 68.99% (P<0.001); and DOP-PCR: 89.09% versus 64.12% (P<0.001)]. In addition, the diagnostic efficiency of ni-PGT in low-quality blastocysts was significantly higher than that in high-quality blastocysts [MALBAC: 95.24% versus 51.85% (P=0.001); and DOP-PCR: 91.30% versus 48.15% (P=0.001)]. These results contribute to further understanding ni-PGT and to clarifying its application scenario in the fresh cycle.

Preimplantation genetic testing for hereditary hearing loss in Chinese population

PurposeTo evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population.MethodsA PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled.ResultsFifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%.Conclusions and relevanceIn both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.

Karyotype of the Blastocoel Fluid Derived by Laser-Assisted Hatching Demonstrates a Low Agreement With the Trophectoderm

ObjectiveThe aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst.MethodsFifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS).ResultsFifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23).ConclusionBlastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised.

Stochastic expression of invasion genes in Plasmodium falciparum schizonts

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental “checkpoint”; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.

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.

Genomic Heterogeneity and Clonal Evolution in Gastroesophageal Junction Cancer Revealed by Single Cell DNA Sequencing.

Gastroesophageal junction (GEJ) cancer is a tumor that occurs at the junction of stomach and esophagus anatomically. GEJ cancer frequently metastasizes to lymph nodes, however the heterogeneity and clonal evolution process are unclear. This study is the first of this kind to use single cell DNA sequencing to determine genomic variations and clonal evolution related to lymph node metastasis. Multiple Annealing and Looping Based Amplification Cycles (MALBAC) and bulk exome sequencing were performed to detect single cell copy number variations (CNVs) and single nucleotide variations (SNVs) respectively. Four GEJ cancer patients were enrolled with two (Pt.3, Pt.4) having metastatic lymph nodes. The most common mutation we found happened in the TTN gene, which was reported to be related with the tumor mutation burden in cancers. Significant intra-patient heterogeneity in SNVs and CNVs were found. We identified the SNV subclonal architecture in each tumor. To study the heterogeneity of CNVs, the single cells were sequenced. The number of subclones in the primary tumor was larger than that in lymph nodes, indicating the heterogeneity of primary site was higher. We observed two patterns of multi-station lymph node metastasis: one was skip metastasis and the other was to follow the lymphatic drainage. Taken together, our single cell genomic analysis has revealed the heterogeneity and clonal evolution in GEJ cancer.

The true incidence of chromosomal mosaicism after preimplantation genetic testing is much lower than that indicated by trophectoderm biopsy.

What is the true incidence of chromosomal mosaicism in embryos analyzed by preimplantation genetic testing (PGT). The true incidence of chromosomal mosaicism is much lower than we usually surmise. 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. 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. 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. 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 < 0.001). 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. 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. 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). N/A.

A Rapid NGS-Based Preimplantation Genetic Testing for Chromosomal Abnormalities in Day-3 Blastomere Biopsy Allows Embryo Transfer Within the Same Treatment Cycle.

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 with de novo abnormalities 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.

Normalized Mitochondrial DNA Copy Number Can Optimize Pregnancy Outcome Prediction in IVF

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.

Comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) in Limited DNA Sequencing Based on Tube and Droplet.

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.

Genome‑wide copy number analysis of circulating tumor cells in breast cancer patients with liver metastasis.

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.

Next-generation sequencing analysis of each blastomere in good-quality embryos: insights into the origins and mechanisms of embryonic aneuploidy in cleavage-stage embryos.

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. 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. 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). 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.

Assessment of two whole genome amplification techniques in terms of soil community profiles

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.

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 3519: High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma

Abstract 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.

Clinical application of NGS-based SNP haplotyping for the preimplantation genetic diagnosis of primary open angle glaucoma

ABSTRACTThe current study describes a successful case of preimplantation genetic diagnosis (PGD) of primary open angle glaucoma (POAG) and verifies the efficiency of next-generation sequencing (NGS)-based haplotyping for PGD of POAG. In this study, we applied NGS as part of PGD to effectively detect POAG prior to embryo implantation and avoid the prospect of pregnancy termination in event of vertical inheritance of POAG. We used the technique of multiple annealing and looping based amplification cycles (MALBAC) to conduct whole genome amplification (WGA) and to reduce the allele dropout (ADO). We also employed Sanger sequencing to directly detect the mutation c.1109 C > T in MYOC and NGS-based single nucleotide polymorphism (SNP) haplotyping to distinguish the chromosomes that carried the mutation. Copy number variation (CNV) analysis was carried out to determine the copy number of embryos’ chromosomes. Of the 4 blastocysts obtained in this study, only 2 (sample 5 and 7) could be successfully amplified by WGA. CNV results indicated that chromosomes of both these samples were balanced (46, XN). Sanger sequencing and NGS-based SNP haplotyping confirmed that sample 7 carried the mutation c.1109 C > T in MYOC, while sample 5 did not. Moreover, no ADO was observed. Thus, blastocyst 5 was transferred into the uterus of the patient, and a healthy baby without the MYOC mutation c. 1109C>T was born 39 weeks after transplantation. Our study suggests that NGS-based SNP haplotyping is an effective technique for the PGD of POAG.Abbreviations: PGD: preimplantation genetic diagnosis; POAG: primary open angle glaucoma; NGS: next-generation sequencing; MALBAC: multiple annealing and looping based amplification cycles chemistry; WGA: whole genome amplification; ADO: allele dropout; SNP: single nucleotide polymorphism; CNV: copy number variation; MYOC: Myocilin; OPTN: Optineurin; WDR36: WD repeat domain 36; CYP1B1: Cytochrome P450 1 B Chain; ICSI: intracytoplasmic sperm injection; TFNA: testicular fine-needle aspiration; TE: trophectoderm; PCR: polymerase chain reaction

Preparing Single-cell DNA Library Using Nextera for Detection of CNV.

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.

A low cost and input tailing method of quality control on multiple annealing, and looping-based amplification cycles-based whole-genome amplification products.

Single-cell whole-genome sequencing provides novel insights into the nature of genetic heterogeneity in normal and diseased cells. However, amplification of formalin-fixed tissues with low cell numbers is still problematic and multiple annealing, and looping-based amplification cycles (MALBAC) is a commonly used whole-genome amplification (WGA) method with low cell numbers. We developed a low-input tailing method to evaluate the MALBAC-based WGA from sub-nanogram or less quantities of input DNA. The tailing method uses 2100 BioAnalyzer to evaluate the size distribution of MALBAC products, and comparing the tailing with 10380bp. Compared with a 22 loci qPCR panel, the tailing method provided a similar WGA evaluation efficiency in 13 samples on one set of study, with lower input, cheaper cost, shorter manual time, and a clear filtering cut off. Later, we demonstrated a strong correlation between tailing size and coverage breadth in another 29 samples on two sets of assays. As a result, the tailing method showed that it could predict whether a sequence breadth achieved 70% or not with 100% accuracy on these three sets of assays. Although further studies are needed, this tailing method is expected to be used as an excellent tool to select high-quality WGA products before library construction. Our tailing method can provide a new WGA quality test to evaluate the WGA efficiency with 100% accuracy (42/42). Compared with qPCR panel, our tailing method needs lower input, cheaper cost, shorter manual time, a clear filtering cut off, and extendable high throughput as well as the same sensitivity.