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\nHIGHLIGHTShort tandem repeat (STR) analysis of a perpetrator’s touch DNA on a victim’s skin is crucial in forensic science. This study evaluates the accuracy of male contributor assignment from skin-to-skin transferred touch DNA using STR profiling with and without whole-genome amplification (WGA). Each of 32 touch DNA samples was collected from a woman’s wrist after being grasped by a man, with each sample divided into three aliquots: one left as a non-WGA sample, another subjected to WGA via multiple displacement amplification (MDA), and the third amplified using modified T oligo-primed PCR (TOP-PCR). Ninety-six (32 × 3) STR profiles were analysed via capillary electrophoresis (CE) and massively parallel sequencing (MPS), with male contributors assigned using EuroForMix. Among the 10 Qubit-detectable samples of the 32 touch DNA samples, the accuracy rates of male contributor assignment using CE were 50% (non-WGA), 40% (MDA) and 70% (TOP-PCR) and using MPS were 100.0% (non-WGA), 50.0% (MDA), and 80.0% (TOP-PCR). For the 22 Qubit-undetectable samples, the accuracy rates using CE were 13.6% (non-WG3A), 4.5% (MDA) and 59.1% (TOP-PCR) and using MPS were 68.2% (non-WGA), 0% (MDA-) and 77.3% (TOP-PCR). In conclusion, TOP-PCR may improve the accuracy of male contributor assignment for Qubit-undetectable samples.WGA using modified TOP-PCR may enhance forensic STR analysis of low-quality touch DNA samples. [ABSTRACT FROM AUTHOR]

Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually

Abstract Objective Environmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed. Results Using fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

Objective: Environmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed. Results: Using fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts. [ABSTRACT FROM AUTHOR]

Introduction: Preimplantation Genetic Testing (PGT) is a cutting-edge test used to detect genetic abnormalities in embryos fertilized through Medically Assisted Reproduction (MAR). PGT aims to ensure that embryos selected for transfer are free of specific genetic conditions or chromosome abnormalities, thereby reducing chances for unsuccessful MAR cycles, complicated pregnancies, and genetic diseases in future children. Areas covered: In PGT, genetics, embryology, and technology progress and evolve together. Biological and technological limitations are described and addressed to highlight complexity and knowledge constraints and draw attention to concerns regarding safety of procedures, clinical validity, and utility, extent of applications and overall ethical implications for future families and society. Expert opinion: Understanding the genetic basis of diseases along with advanced technologies applied in embryology and genetics contribute to faster, cost-effective, and more efficient PGT. Next Generation Sequencing-based techniques, enhanced by improved bioinformatics, are expected to upgrade diagnostic accuracy. Complicating findings such as mosaicism, mt-DNA variants, variants of unknown significance, or variants related to late-onset or polygenic diseases will however need further appraisal. Emphasis on monitoring such emerging data is crucial for evidence-based counseling while standardized protocols and guidelines are essential to ensure clinical value and respect of Ethical, Legal and Societal Issues. [ABSTRACT FROM AUTHOR]

Abstract Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights. [ABSTRACT FROM AUTHOR]

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention. [ABSTRACT FROM AUTHOR]

Objective: To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Design: Article. Patient(s): fifty-two deletional α-thalassemia couples. Intervention(s): Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. Main outcome measures: WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments. [ABSTRACT FROM AUTHOR]

Scientific drilling expeditions offer a unique opportunity to characterize the microbial communities in the subsurface that have been long-term isolated from the surface. With subsurface microbial biomass being low in general, biological contamination from the drilling fluid, sample processing, or molecular work is a major concern. To address this, characterization of the contaminant populations in the drilling fluid and negative extraction controls are essential for assessing and evaluating such sequencing data. Here, crystalline rock cores down to 2250 m depth, groundwater-bearing fractures, and the drilling fluid were sampled for DNA to characterize the microbial communities using a broad genomic approach. However, even after removing potential contaminant populations present in the drilling fluid, notorious contaminants were abundant and mainly affiliated with the bacterial order Burkholderiales. These contaminant microorganisms likely originated from the reagents used for isolating and amplifying DNA despite stringent quality standards during the molecular work. The detection of strictly anaerobic sulfate reducers such as Candidatus Desulforudis audaxviator suggested the presence of autochthonous deep biosphere taxa in the sequenced libraries, yet these clades represented only a minor fraction of the sequence counts (< 0.1 %), hindering further ecological interpretations. The described methods and findings emphasize the importance of sequencing extraction controls and can support experimental design for future microbiological studies in conjunction with continental drilling operations. [ABSTRACT FROM AUTHOR]

Multiple displacement amplification (MDA) based on isothermal random priming and high fidelity phi29 DNA polymerase-mediated processive extension has revolutionized the field of whole genome amplification by enabling the amplification of minute amounts of DNA, such as from a single cell, generating vast amounts of DNA with high genome coverage. Despite its advantages, MDA has its own challenges, one of the grandest being the formation of chimeric sequences (chimeras), which presents in all MDA products and seriously disturbs the downstream analysis. In this review, we provide a comprehensive overview of current research on MDA chimeras. We first reviewed the mechanisms of chimera formation and chimera detection methods. We then systematically summarized the characteristics of chimeras, including overlap, chimeric distance, chimeric density, and chimeric rate, as found in independently published sequencing data. Finally, we reviewed the methods used to process chimeric sequences and their impacts on the improvement of data utilization efficiency. The information presented in this review will be useful for those interested in understanding the challenges with MDA and in improving its performance.

Purpose: To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. Methods: A 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. Results: Fifty-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 relevance: In 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. [ABSTRACT FROM AUTHOR]

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity. [Display omitted] • SoNIC is an ideal method for single cardiac nuclei isolation from human postmortem tissue for WGA and RNAseq. • Ploidy-based single-nuclei sorting provides a pure cardiomyocyte nuclei isolation strategy. • A negative correlation between DIN and PMI or age suggests the inclusion of DIN along with RIN for tissue quality measures. • DIN over 5.8 could be used for WGA and RNAseq analysis without sacrificing key signals for biological investigation. [ABSTRACT FROM AUTHOR]

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions. [ABSTRACT FROM AUTHOR]

Purpose of reviewThe purpose of this review is to highlight recent research advances in noninvasive prenatal diagnostic methods.Recent findingsRecent studies developing noninvasive prenatal diagnostic (NIPD) methods have been focused on either fetal nucleated red blood cells (fNRBCs) or circulating trophoblasts (cTBs). Enriched cTBs were successfully utilized for whole genome profiling and short tandem repeat (STR) identification to confirm feto-maternal relationship. However, further analysis of isolated fNRBCs remains confined to examining fetal cytogenetics.SummaryInvasive prenatal diagnostic procedures, amniocentesis and chorionic villus sampling, are the gold standard for the diagnosis of fetal chromosomal abnormalities and genetic disorders. Meanwhile, noninvasive techniques of analyzing circulating cell-free fetal DNA (cffDNA) have been limited to screening tools and are highly fragmented and confounded by maternal DNA. By detecting circulating fetal nucleated cells (CFNCs) we are able to noninvasively confirm fetal chromosomal abnormalities, truly realizing the concept of "noninvasive prenatal diagnostics". The primary technical challenge is the enrichment of the low abundance of CFNCs in maternal peripheral blood. For any cell-based NIPD method, both fetal whole genome profiling and confirmation of the feto-parental relationship are essential. This has been successfully performed using enriched and isolated cTBs, making cTB a better candidate for NIPD. cTB enumeration also correlates with abnormal fetal or placental development. On the other hand, downstream analysis of fNRBCs remains limited to examining fetal sex and aneuploidies. Furthermore, trophoblast-based NIPD via an endocervical sample is also promising because of reduced dilution from hematologic cells.

Formalin‐fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre‐amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde‐damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi‐allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin‐fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP‐based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage. [ABSTRACT FROM AUTHOR]

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment. [ABSTRACT FROM AUTHOR]

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

Abstract Background Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). Results In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. Conclusion The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.

Background: Genomic profiling in lung cancer is essential for precision medicine. Cytological specimens provide an alternative to formalin‐fixed paraffin‐embedded (FFPE) samples for comprehensive genomic analysis. However, this approach remains challenging when a limited number of tumor cells are available. We applied whole genome amplification (WGA) to cytology specimens to overcome this limitation. Methods: Using a lung cancer panel targeting 58 genes, we performed next‐generation sequencing of whole genome‐amplified DNA extracted from cytological specimens containing 10–20 tumor cells (cyto‐WGA) and DNA from corresponding FFPE tumor tissue. We compared sequencing data from cyto‐WGA and FFPE samples to examine the detection accuracy of copy number variations and oncogenic and drug‐matched variants. Results: The DNA quality and quantity from cyto‐WGA were higher than those from FFPE samples (p <.0005 and p <.05, respectively). Sequencing metrics of cyto‐WGA and FFPE tissues showed no difference in the number of mapped reads and mean coverage depth, but there were significant differences in the on‐target rate (p <.05) and uniformity (p <.0005). Copy number variations in cyto‐WGA samples (n = 211) were higher than in FFPE samples (n = 9) (p <.0001). Fourty nine oncogenic variants were detected in cyto‐WGA and 39 in FFPE. Of these variants, 34 (63%) were present in both samples. In addition, all 16 drug‐matched variants were detected in FFPE and cyto‐WGA samples with 100% concordance. Conclusion: Cyto‐WGA can be a feasible and alternative method to detect oncogenic and drug‐matched variants. The authors compared sequencing data from cytological whole genome amplification (WGA) and formalin‐fixed paraffin‐embedded samples to examine the detection accuracy of copy number variations and oncogenic and drug‐matched variants. Cytological WGA can be a feasible and alternative method to detect oncogenic and drug‐matched variants. [ABSTRACT FROM AUTHOR]

The phylum Nematoda represents one of the most cosmopolitan and abundant metazoan groups on Earth. In this study, we reconstructed the phylogenomic tree for phylum Nematoda. A total of 60 genomes, belonging to eight nematode orders, were newly sequenced, providing the first low-coverage genomes for the orders Dorylaimida, Mononchida, Monhysterida, Chromadorida, Triplonchida, and Enoplida. The resulting phylogeny is well-resolved across most clades, with topologies remaining consistent across various reconstruction parameters. The subclass Enoplia is placed as a sister group to the rest of Nematoda, agreeing with previous published phylogenies. While the order Triplonchida is monophyletic, it is not well-supported, and the order Enoplida is paraphyletic. Taxa possessing a stomatostylet form a monophyletic group; however, the superfamily Aphelenchoidea does not constitute a monophyletic clade. The genera Trichinella and Trichuris are inferred to have shared a common ancestor approximately 202 millions of years ago (Ma), a considerably later period than previously suggested. All stomatostylet-bearing nematodes are proposed to have originated ~305 Ma, corresponding to the transition from the Devonian to the Permian period. The genus Thornia is placed outside of Dorylaimina and Nygolaimina, disagreeing with its position in previous studies. Additionally, we tested the whole genome amplification method and demonstrated that it is a promising strategy for obtaining sufficient DNA for phylogenomic studies of microscopic eukaryotes. This study significantly expanded the current nematode genome dataset, and the well-resolved phylogeny enhances our understanding of the evolution of Nematoda., (© The Author(s) 2024. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Y chromosome plays an important role in the sexual reproduction of many species because it contains sex-determining genes and male reproductive factors and the Y chromosome has only been sequenced in a few mammalian species. Y chromosome has been study in several fields, such as spermatogenesis, male development and sex chromosome evolution. The giant panda (Ailuropoda melanoleuca) is an endangered species in China. Male giant pandas have reproductive defects such as low natural mating rate, poor sperm quality, and infertility that inhibit population growth. More information is needed on the genetics of giant panda reproduction in order to understand their biological characteristics. The Y chromosome data of the giant panda are now unavailable, though a draft genome of this species has been sequenced. Thus, in the present study, we separated single Y chromosomes of male giant panda, sequenced using high-throughput sequencing after whole genome amplification. Finally, two genes, ZFY and TSPY1, on Y chromosome and related to male fertility were annotated. This study is a supplement of giant panda genetic data. [ABSTRACT FROM AUTHOR]

BackgroundCancer research to date has largely focused on somatically acquired genetic aberrations. In contrast, the degree to which germline, or inherited, variation contributes to tumorigenesis remains unclear, possibly due to a lack of accessible germline variant data. Here we called germline variants on 9618 cases from The Cancer Genome Atlas (TCGA) database representing 31 cancer types.ResultsWe identified batch effects affecting loss of function (LOF) variant calls that can be traced back to differences in the way the sequence data were generated both within and across cancer types. Overall, LOF indel calls were more sensitive to technical artifacts than LOF Single Nucleotide Variant (SNV) calls. In particular, whole genome amplification of DNA prior to sequencing led to an artificially increased burden of LOF indel calls, which confounded association analyses relating germline variants to tumor type despite stringent indel filtering strategies. The samples affected by these technical artifacts include all acute myeloid leukemia and practically all ovarian cancer samples.ConclusionsWe demonstrate how technical artifacts induced by whole genome amplification of DNA can lead to false positive germline-tumor type associations and suggest TCGA whole genome amplified samples be used with caution. This study draws attention to the need to be sensitive to problems associated with a lack of uniformity in data generation in TCGA data.

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies. [ABSTRACT FROM AUTHOR]

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called "liquid biopsies". Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection. [ABSTRACT FROM AUTHOR]

Single cell genomics offers an unprecedented resolution to interrogate genetic heterogeneity in a patient’s tumour at the intercellular level. However, the DNA yield per cell is insufficient for today’s sequencing library preparation protocols. This necessitates DNA amplification which is a key source of experimental noise. We provide an evaluation of two protocols using micro-fluidics based amplification for whole exome sequencing, which is an experimental scenario commonly used in single cell genomics. The results highlight their respective biases and relative strengths in identification of single nucleotide variations. Towards this end, we introduce a workflow SoVaTSiC, which allows for quality evaluation and somatic variant identification of single cell data. As proof of concept, the framework was applied to study a lung adenocarcinoma tumour. The analysis provides insights into tumour phylogeny by identifying key mutational events in lung adenocarcinoma evolution. The consequence of this inference is supported by the histology of the tumour and demonstrates usefulness of the approach.

Background: Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). Results: In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. Conclusion: The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome. [ABSTRACT FROM AUTHOR]

Whole genome amplification (WGA) is a technology for non‐selective amplification of the whole genome sequence, first appearing in 1992. Its primary purpose is to amplify and reflect the whole genome of trace tissues and single cells without sequence bias and to provide sufficient DNA template for subsequent multigene and multilocus analysis, along with comprehensive genome research. WGA provides a method to obtain a large amount of genetic information from a small amount of DNA and provides a valuable tool for preserving limited samples in molecular biology. WGA technology is especially suitable for forensic identification and genetic disease research, along with new technologies such as next‐generation sequencing (NGS). In addition, WGA is also widely used in single‐cell sequencing. Due to the small amount of DNA in a single cell, it is often unable to meet the amount of samples needed for sequencing, so WGA is generally used to achieve the amplification of trace samples. This paper reviews WGA methods based on different principles, summarizes both amplification principle and amplification quality, and discusses the application prospects and challenges of WGA technology in molecular diagnosis and medicine. [ABSTRACT FROM AUTHOR]

Abstract Background The study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA. However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection. Results We propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method. Conclusion Scellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.

Abstract Background Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated. Methods Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g®, MALBACTM, PicoPLEX®, and GenomePlex®, for which DNA quantity and quality were compared between original DNA and post-WGA products. Results The proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value

Abstract Whole genome amplification (WGA) is a technology for non‐selective amplification of the whole genome sequence, first appearing in 1992. Its primary purpose is to amplify and reflect the whole genome of trace tissues and single cells without sequence bias and to provide sufficient DNA template for subsequent multigene and multilocus analysis, along with comprehensive genome research. WGA provides a method to obtain a large amount of genetic information from a small amount of DNA and provides a valuable tool for preserving limited samples in molecular biology. WGA technology is especially suitable for forensic identification and genetic disease research, along with new technologies such as next‐generation sequencing (NGS). In addition, WGA is also widely used in single‐cell sequencing. Due to the small amount of DNA in a single cell, it is often unable to meet the amount of samples needed for sequencing, so WGA is generally used to achieve the amplification of trace samples. This paper reviews WGA methods based on different principles, summarizes both amplification principle and amplification quality, and discusses the application prospects and challenges of WGA technology in molecular diagnosis and medicine.

Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analyzing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.

Blood samples have traditionally been used as the main source of DNA for genetic analysis. However, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood samples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.

Objective To evaluate the efficiency of REPLI-g?? Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g?? Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. GoldeneyeTM DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g?? Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony?? DNA Investigator?? Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g?? Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g?? Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution. [ABSTRACT FROM AUTHOR]

In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from “Eureka” lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid (“CSO”). Data obtained from the “CSO” pollen nuclei population were compared to those obtained from genotyping of a segregating population (“RTSO”) arising from a hand-made sexual hybridization between diploid non apomictic selected tangor (mandarin × sweet orange; “RTO” tangor) as female parent pollinated with “CSO” tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five “Eureka” lemon pollen nuclei. The rate of SD observed in “CSO” pollen nuclei (13.8%) was slightly lower than that recovered in the “RTSO” population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the “RTSO” population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.

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

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.

Abstract Background The recommended genomic DNA input requirements for whole genome single nucleotide polymorphism microarrays can limit the scope of molecular epidemiological studies. We performed a large-scale evaluation of whole genome amplified DNA as input into high-density, whole-genome Illumina® Infinium® SNP microarray. Results Overall, 6622 DNA samples from 5970 individuals were obtained from three distinct biospecimen sources and genotyped using gDNA and/or wgaDNA inputs. When genotypes from the same individual were compared with standard, native gDNA input amount, we observed 99.94% mean concordance with wgaDNA input. Conclusions Our results demonstrate that carefully conducted studies with wgaDNA inputs can yield high-quality genotyping results. These findings should enable investigators to consider expansion of ongoing studies using high-density SNP microarrays, currently challenged by small amounts of available DNA.