Bulk DNA Samples Research Articles

Evaluating zooplankton species diversity using environmental DNA and bulk-DNA metabarcoding in the Ulleung Basin of the Southeastern Korean Peninsula in the summer

Accurately investigating the composition of zooplankton species is crucial for monitoring changes in marine ecosystems and assessing biodiversity. In this study, we utilized bulk DNA and environmental DNA metabarcoding in the Ulleung Basin, known for its high zooplankton species diversity among the seas surrounding the Korean Peninsula. Genomic DNA extracted from samples collected during three summer seasons in the survey area was analyzed using high-throughput sequencing of the cytochrome c oxidase I barcode region. We identified 350 species, which were three to six times more than those identified by traditional morphological methods. Furthermore, we observed significant differences in species composition and diversity between bulk DNA and eDNA samples. Notably, eDNA metabarcoding effectively detected species with high swimming ability and those that were difficult to capture using traditional sampling methods. This study underscores the significant impact of sampling methods on research outcomes in zooplankton species diversity studies and highlights the importance of integrating different sampling techniques. Specifically, it suggests the need for the active adoption of non-invasive methods, such as eDNA metabarcoding, for the comprehensive monitoring of diverse biological groups in marine ecosystems.

Unlocking rivers' hidden diversity and ecological status using DNA metabarcoding in Northwest Spain.

Rivers are crucial ecosystems supporting biodiversity and human well-being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi-tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems.

From DNA to diagnostics: A case study using macroinvertebrate metabarcoding to assess the effectiveness of restoration measures in a Dutch stream

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers.

COI metabarcoding of large benthic Foraminifera: Method validation for application in ecological studies.

Monitoring community composition of Foraminifera (single-celled marine protists) provides valuable insights into environmental conditions in marine ecosystems. Despite the efficiency of environmental DNA (eDNA) and bulk-sample DNA (bulk-DNA) metabarcoding to assess the presence of multiple taxa, this has not been straightforward for Foraminifera partially due to the high genetic variability in widely used ribosomal markers. Here, we test the correctness in retrieving foraminiferal communities by metabarcoding of mock communities, bulk-DNA from coral reef sediment samples, and eDNA from their associated ethanol preservative using the recently sequenced cytochrome c oxidase subunit 1 (COI) marker. To assess the detection success, we compared our results with large benthic foraminiferal communities previously reported from the same sampling sites. Results from our mock communities demonstrate that all species were detected in two mock communities and all but one in the remaining four. Technical replicates were highly similar in number of reads for each assigned ASV in both the mock communities and bulk-DNA samples. Bulk-DNA showed a significantly higher species richness than their associated eDNA samples, and also detected additional species to what was already reported at the specific sites. Our study confirms that metabarcoding using the foraminiferal COI marker adequately retrieves the diversity and community composition of both the mock communities and the bulk-DNA samples. With its decreased variability compared with the commonly used nuclear 18 S rRNA, the COI marker renders bulk-DNA metabarcoding a powerful tool to assess foraminiferal community composition under the condition that the reference database is adequate to the target taxa.

Embryo tracking system for high-throughput sequencing-based preimplantation genetic testing

Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)? Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially. Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach. We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS. We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined. Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods. Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT. The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing. M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. N/A.

Efficient low-cost marker-assisted selection of trees with MALE STERILITY 1 (MS1) in Japanese cedar (Cryptomeria japonica D. Don) using bulk DNA samples

Efficient low-cost marker-assisted selection of trees with MALE STERILITY 1 (MS1) in Japanese cedar (Cryptomeria japonica D. Don) using bulk DNA samples

Reconstructing Complex Cancer Evolutionary Histories from Multiple Bulk DNA Samples Using Pairtree.

Clone trees illustrate the evolutionary history of a cancer and can provide insights into how the disease changed through time (e.g., between diagnosis and relapse). Pairtree uses DNA-sequencing data from many samples of the same cancer to build more detailed and accurate clone trees than previously possible. See related commentary by Miller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples.

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples.

Detection of Macrobenthos Species With Metabarcoding Is Consistent in Bulk DNA but Dependent on Body Size and Sclerotization in eDNA From the Ethanol Preservative

DNA metabarcoding is a promising method to increase cost and time efficiency of marine monitoring. While substantial evidence exists that bulk DNA samples adequately reflect diversity patterns of marine macrobenthos, the potential of eDNA in the ethanol preservative of benthic samples for biodiversity monitoring remains largely unexplored. We investigated species detection in bulk DNA and eDNA from the ethanol preservative in samples from four distinct macrobenthic communities in the North Sea. Bulk DNA and eDNA were extracted with different extraction kits and five COI primer sets were tested. Despite the availability of a nearly complete reference database, at most 22% of the amplicon sequence variants (ASVs) were assigned taxonomy at the phylum level. However, the unassigned ASVs represented only a small fraction of the total reads (13%). The Leray primer set outperformed the four other primer sets in the number of non-chimeric reads and species detected, and in the recovery of beta diversity patterns. Community composition differed significantly between bulk DNA and eDNA samples, but both sample types were able to differentiate the four communities. The probability of detecting a species in the eDNA from the ethanol preservative was significantly lower than for bulk DNA for macrobenthos species having small to medium body size and for species having chitine or CaCO3 in their cuticula. Detection in the bulk DNA samples was not affected by the investigated morphological traits, indicating that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

Comprehensive identification of somatic nucleotide variants in human brain tissue

BackgroundPost-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells.ResultsHere, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees.ConclusionsThis study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

To blend or not to blend? The role of morphological traits for the detection of marine macrobenthos in bulk DNA and eDNA from the ethanol preservative

The impact of methodological choices on the reliability and reproducibility of DNA metabarcoding need to be well understood to allow successful implementation in routine monitoring frameworks. For macrobenthos communities, the metabarcoding protocol focuses on a fragment of the mitochondrial COI gene and depending on the primer set used for amplification of COI, different taxa can be detected. To identify the primer set that allows the best diversity estimates for macrobenthos in the North Sea region, we sampled four distinct and well characterised communities and identified macrobenthos using traditional morpho-taxonomy before molecular processing. Of the five primer sets tested, the Leray primer set yielded the highest number of non-chimeric reads, detected the highest number of macrobenthos species and best recovered beta diversity patterns. Despite the availability of a nearly complete reference database, 19 out of the 59 morphological species were not picked up with DNA metabarcoding. Next to primer choice, the DNA source used in metabarcoding studies can affect whether or not a species is detected. DNA can be extracted from bulk specimens or from the ethanol preservative in which the macrobenthos sample was preserved. The latter DNA source would greatly speed up processing time of samples in the laboratory. We therefore compared species detection in bulk DNA and eDNA from the ethanol preservative from the four macrobenthos communities in the North Sea. Our results show that community composition differed significantly between bulk DNA and eDNA samples, but both sample types are able to differentiate the four macrobenthos communities from the North Sea. Of the 49 species that are detected in both sample types, 27 are also found in the morphological dataset. The 14 species that are exclusively detected in the ethanol preservative are mainly pelagic species. In view of the low read numbers allocated to these species (at most 153 reads) they most likely represent “contaminant” DNA molecules that are attached to the specimens or the organic debris. To better understand the different results between bulk DNA and eDNA from the ethanol preservative, we investigated the importance of four categorical traits in explaining the probability of detecting a species in the two sample types: body, larval stage (benthic or pelagic), longevity and body skeleton (chitin, CaCO3 or soft tissue). A generalized linear mixed effects model approach shows that the probability of detecting a species in the eDNA from the ethanol preservative is significantly lower than for bulk DNA for macrobenthos species having small to medium body size and for species having chitine or CaCO3 in their skeleton. In contrast, detection in the bulk DNA samples is not affected by the investigated traits. Although the ethanol preservative can be used to characterize beta diversity patterns, our results show that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding.

Kinship analysis on single cells after whole genome amplification

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and pre-implantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STR- and SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using high-quality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results.

Capturing open ocean biodiversity: Comparing environmental DNA metabarcoding to the continuous plankton recorder.

Environmental DNA (eDNA) metabarcoding is emerging as a novel, objective tool for monitoring marine metazoan biodiversity. Zooplankton biodiversity in the vast open ocean is currently monitored through continuous plankton recorder (CPR) surveys, using ship-based bulk plankton sampling and morphological identification. We assessed whether eDNA metabarcoding (2L filtered seawater) could capture similar Southern Ocean zooplankton biodiversity as conventional CPR bulk sampling (~1,500L filtered seawater per CPR sample). We directly compared eDNA metabarcoding with (a) conventional morphological CPR sampling and (b) bulk DNA metabarcoding of CPR collected plankton (two transects for each comparison, 40 and 44 paired samples, respectively). A metazoan-targeted cytochrome c oxidase I (COI) marker was used to characterize species-level diversity. In the 2L seawater eDNA samples, this marker amplified large amounts of non-metazoan picoplanktonic algae, but eDNA metabarcoding still detected up to 1.6 times more zooplankton species than morphologically analysed bulk CPR samples. COI metabarcoding of bulk DNA samples mostly avoided nonmetazoan amplifications and recovered more zooplankton species than eDNA metabarcoding. However, eDNA metabarcoding detected roughly two thirds of metazoan species and identified similar taxa contributing to community differentiation across the subtropical front separating transects. We observed a diurnal pattern in eDNA data for copepods which perform diel vertical migrations, indicating a surprisingly short temporal eDNA signal. Compared to COI, a eukaryote-targeted 18S ribosomal RNA marker detected a higher proportion, but lower diversity, of metazoans in eDNA. With refinement and standardization of methodology, eDNA metabarcoding could become an efficient tool for monitoring open ocean biodiversity.

Abstract 4969: Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR

Abstract The absolute quantification of adaptive immune cells in cancer is of clinical importance for diagnosis, prognosis and treatment. First, the cancer itself may be of lymphoproliferative origin, in which an accurate cell count facilitates the diagnosis. Alternatively, healthy T- and B-lymphocytes may be infiltrating a tumor, affecting tumor progression or representing a potential therapeutic target. However, sample quantity and quality do not always allow to perform cell- or tissue-based quantitative analyses. As an alternative, we identified generic DNA markers of a variety of adaptive immune cell types. These markers were translated into a digital PCR experimental setup, enabling the absolute quantification of these cell types in bulk DNA. The sensitivity and dynamic range of this approach were technically validated in control samples and calibration curves. Moreover, a biological validation showed a precision of our measurements comparable to flow cytometry (R2 > 0.9). Importantly, as our sample requirements are much lower (5-20 ng of DNA instead of intact cells), this approach provides a novel, sensitive way for the accurate quantification of adaptive immune cells independent of the cellular context. We evaluated our approach in the context of two clinical applications: diagnosis of vitreoretinal lymphoma by a liquid biopsy, and absolute deconvolution of the immune infiltrate in melanoma. Vitreoretinal lymphoma is a clinically challenging masquerade syndrome, frequently mimicking a benign uveitis. To confirm the diagnosis, a vitreous biopsy may be obtained and analyzed for malignant cells. However, analysis may be impeded by low cellularity or loss of cellular integrity. With our approach, we could acquire absolute immune cell quantifications from only 10 uL vitreous fluid, independent of cellular context. To analyze the immune infiltrate from bulk DNA or RNA samples in solid tumors, numerous deconvolution approaches are available. However, these methods typically rely on relative differences in gene expression, consequently lacking the precision to be compared with absolute measurements. Our DNA-based method allows to obtain absolute lymphocyte quantifications, comparable to (single) cell-based technologies. In both cutaneous and uveal melanoma, this facilitated an enhanced deconvolution of the bulk expression analysis. In conclusion, we developed and validated a digital PCR-based approach to quantify immune cells in bulk DNA samples. A similar precision could be obtained compared to flow cytometry, but with much lower requirements concerning sample quality and quantity. Therefore, this method can be broadly applied in a wide range of sample types, supporting cancer diagnosis, prognosis and treatment. Citation Format: Rogier J. Nell, Mieke Versluis, Willem H. Zoutman, Pieter A. Van der Velden. Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4969.

Implications of non-uniqueness in phylogenetic deconvolution of bulk DNA samples of tumors

BackgroundTumors exhibit extensive intra-tumor heterogeneity, the presence of groups of cellular populations with distinct sets of somatic mutations. This heterogeneity is the result of an evolutionary process, described by a phylogenetic tree. In addition to enabling clinicians to devise patient-specific treatment plans, phylogenetic trees of tumors enable researchers to decipher the mechanisms of tumorigenesis and metastasis. However, the problem of reconstructing a phylogenetic tree T given bulk sequencing data from a tumor is more complicated than the classic phylogeny inference problem. Rather than observing the leaves of T directly, we are given mutation frequencies that are the result of mixtures of the leaves of T. The majority of current tumor phylogeny inference methods employ the perfect phylogeny evolutionary model. The underlying Perfect Phylogeny Mixture (PPM) combinatorial problem typically has multiple solutions.ResultsWe prove that determining the exact number of solutions to the PPM problem is #P-complete and hard to approximate within a constant factor. Moreover, we show that sampling solutions uniformly at random is hard as well. On the positive side, we provide a polynomial-time computable upper bound on the number of solutions and introduce a simple rejection-sampling based scheme that works well for small instances. Using simulated and real data, we identify factors that contribute to and counteract non-uniqueness of solutions. In addition, we study the sampling performance of current methods, identifying significant biases.ConclusionsAwareness of non-uniqueness of solutions to the PPM problem is key to drawing accurate conclusions in downstream analyses based on tumor phylogenies. This work provides the theoretical foundations for non-uniqueness of solutions in tumor phylogeny inference from bulk DNA samples.

Agilent Technologies OnePGT solution: External verification on both blastomere and trophectoderm biopsies

Objectives OnePGT is a genome-wide next-generation sequencing haplarithmisis-based solution designed to reinforce ranking of IVF embryos. The single workflow solution, consisting of wet-lab reagents and dedicated data analysis software, allows concurrent PGT-M (preimplantation genetic testing of monogenic disorders), PGT-SR (preimplantation genetic testing of structural rearrangements) and PGT-A (preimplantation genetic testing of aneuploidies) hence enabling enhanced genetic profiling. In collaboration with MUMC and KU Leuven, a study was set up to assess the level of concordance between OnePGT and Gold Standard analysis of both single and few cell embryo biopsies for PGT-M, PGT-SR and PGT-A. Methods A total of 227 embryo samples (191 PGT-M; 36 PGT-SR) were included in the study, consisting of 179 blastomere and 48 trophectoderm biopsies, of which 118 embryos were obtained from KU Leuven, and 109 from MUMC. For the KU Leuven site of the study, left-over whole genome amplified (WGAed) DNA from the embryo biopsies was processed, while in the MUMC site, a second biopsy was taken and WGAed. Libraries for NGS were generated using proprietary OnePGT reagents for all WGAed embryo biopsies and for bulk DNA samples from parents and reference family member(s) in case of PGT-M. Libraries were sequenced on NextSeq500 (Illumina) and resulting data were analyzed with the dedicated proprietary OnePGT software suite. Results For PGT-M analysis, a OnePGT conclusion was made for 91 % of embryos determined to be of sufficient quality (n=161). Of these, four were shown to have mitotic aberrations resulting from chromosome instability phenomenon and three did not receive a conclusion from the Gold Standard. The remaining 154 embryos showed 100% concordance with the standard PGT techniques. For PGT-SR analysis (n=36), three embryos were shown to have mitotic aberrations resulted from chromosome instability phenomenon, the remaining 33 embryos 100% concordance with the standard PGT methods. For PGT-A analysis, 100% concordance (> 50 Mb) was shown between OnePGT and standard PGT methods for which a PGT-A result was available (n=118). Conclusion The proprietary OnePGT solution, consisting of all-in-one library preparation reagents and dedicated software suite for concurrent PGT-M, PGT-SR and PGT-A in a single assay, successfully identified the presence of 26 different monogenic disorders, translocations and chromosomal aneuploidies in over 200 embryo biopsies, with a concordance rate of 100%. This indicates that this innovative genome-wide solution is expected to boost the current PGT practice and to facilitate effective ranking of IVF embryos based on enhanced genetic profiling.

Detection of Heteroplasmic Variants in the Mitochondrial Genome through Massive Parallel Sequencing.

Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled pipeline is necessary to reliably retrieve and quantify the low frequency variants. It has been shown that differences in methods can significantly affect the number and frequency of the retrieved variants. In this protocol, we present a method involving both wet lab and bioinformatics that allows identifying and quantifying single nucleotide variants in the full mtDNA sequence, down to a heteroplasmic load of 1.5%. For this, we set up a PCR-based amplification of the mtDNA, followed by MPS using Illumina chemistry, and variant calling with two different algorithms, mtDNA server and Mutect. The PCR amplification is used to enrich the mitochondrial fraction, while the bioinformatic processing with two algorithms is used to discriminate the true heteroplasmies from background noise. The protocol described here allows for deep sequencing of the mitochondrial DNA in bulk DNA samples as well as single cells (both large cells such as human oocytes, and small-sized single cells such as human embryonic stem cells) with minor modifications to the protocol.

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy.

DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectron and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. Both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.

Genetic characterization of SF3B1 mutations in single chronic lymphocytic leukemia cells

Genetic characterization of SF3B1 mutations in single chronic lymphocytic leukemia cells

MARCADORES RAPDs ASOCIADOS A LA EXPRESIÓN DEL SEXO EN Ceratozamia mexicana Brongniart (Zamiaceae)

Ceratozamia mexicana Brongniart is a strictly dioecious species with a long juvenile stage. In order to detect molecular markers associated with sex, it was analyzed by the technique of random amplified polymorphic DNA (RAPD) and masal segregant analysis (BSA) samples of leaf tissue of leaf tissue of sexually differentiated individuals of the population of this species located at Coacoatzintla, Veracruz, México. 5 primers were used to amplify three bulk DNA samples of five known individuals from each sex. A total of 37 bands were produced of which 63% were polymorphic. The OPB08 and OPK10 primers generated distinctive bands to male sex in C. mexicana. With the OPK10 primer a band of 500 bp was observed in two of the three male bulk samples tested, while the OPB08 primer detected two bands (1400 and 1100 bp) in the male samples tested. Although it is necessary to validate this observation, these results could open a new alternative to early determination of sex in C. mexicana.