DNA Sequencing Methods Research Articles

EP20.43: The rapid long read DNA sequencing method identified bacterial species causing silent intra‐amniotic infection

EP20.43: The rapid long read DNA sequencing method identified bacterial species causing silent intra‐amniotic infection

Blast from the past: a study of decades-old fungal cultures resolves a long-standing tree disease mystery

A root disease in plantations of Pinus radiata and Pinus pinaster, where trees died in distinct patches, was present in the Western Cape province of South Africa during the 1970s and 1980s. Phytophthora cinnamomi was initially believed to be the cause, but the disease was later ascribed to the insect-associated fungus Leptographium serpens, a fungal species residing in the Ophiostomatales. Doubt regarding the cause of the disease was raised in a later study due to the fact that most Leptographium spp., particularly those that colonise ray parenchyma tissues, which is the case for L. serpens, are not typically primary disease agents. In this study, cultures of an unidentified sterile fungus collected from the dying trees were revived and identified using DNA sequencing methods, which were not available when the disease was first studied. These cultures were identified as the pyrophillic pathogen Rhizina undulata, well-known to cause patch death of conifers in South Africa and elsewhere in the world. While the patches of dying trees no longer exist and the disease cannot be newly studied, it is most likely that the tree death originally thought to be caused by L. serpens was due primarily to R. undulata. The study provides a vivid example of the value of preserving cultures of fungi for later study and the power of modern techniques to identify fungal pathogens.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only “canonical” mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.

Exploration of pathogenic microorganism within the small intestine of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common severe gastrointestinal emergency in neonates. We designed this study to identify the pathogenic microorganisms of NEC in the microbiota of the small intestine of neonates. Using the 16S ribosomal DNA (rDNA) sequencing method, we compared and analyzed the structure and diversity of microbiotas in the intestinal feces of different groups of neonates: patients undergoing jejunostomy to treat NEC (NP group), neonates undergoing jejunostomy to treat other conditions (NN group), and neonates with NEC undergoing conservative treatment (NC group). We took intestinal feces and saliva samples from patients at different time points. The beta diversities of the NP, NN, and NC groups were all similar. When comparing the beta diversities between different time points in the NP group, we found similar beta diversities at time points E1 to E3 but significant differences between the E2-E3 and E4 time points: the abundances of Klebsiella and Enterococcus (Proteobacteria) were higher at the E1-E3 time points; the abundance of Escherichia-Shigella (Proteobacteria) increased at the E2 time point, and the abundance of Klebsiella decreased significantly, whereas that of Streptococcus increased significantly at the E4 time point. Our results suggest that the pathological changes of intestinal necrosis in the small intestine of infants with NEC are not directly caused by excessive proliferation of pathogenic bacteria in the small intestine. The sources of microbiota in the small intestine of neonates, especially in premature infants, may be affected by multiple factors.

Scaling-up RADseq methods for large datasets of non-invasive samples: Lessons for library construction and data preprocessing.

Genetic non-invasive sampling (gNIS) is a critical tool for population genetics studies, supporting conservation efforts while imposing minimal impacts on wildlife. However, gNIS often presents variable levels of DNA degradation and non-endogenous contamination, which can incur considerable processing costs. Furthermore, the use of restriction-site-associated DNA sequencing methods (RADseq) for assessing thousands of genetic markers introduces the challenge of obtaining large sets of shared loci with similar coverage across multiple individuals. Here, we present an approach to handling large-scale gNIS-based datasets using data from the spotted hyena population inhabiting the Ngorongoro Crater in Tanzania. We generated 3RADseq data for more than a thousand individuals, mostly from faecal mucus samples collected non-invasively and varying in DNA degradation and contamination level. Using small-scale sequencing, we screened samples for endogenous DNA content, removed highly contaminated samples, confirmed overlap fragment length between libraries, and balanced individual representation in a sequencing pool. We evaluated the impact of (1) DNA degradation and contamination of non-invasive samples, (2) PCR duplicates and (3) different SNP filters on genotype accuracy based on Mendelian error estimated for parent-offspring trio datasets. Our results showed that when balanced for sequencing depth, contaminated samples presented similar genotype error rates to those of non-contaminated samples. We also showed that PCR duplicates and different SNP filters impact genotype accuracy. In summary, we showed the potential of using gNIS for large-scale genetic monitoring based on SNPs and demonstrated how to improve control over library preparation by using a weighted re-pooling strategy that considers the endogenous DNA content.

Identification of a novel variant c.761C>T on ABO*B.01 gene in ABO glycosyltransferases associated with Bweak phenotype.

ABO antigens are produced from H antigen by the activity of glycosyltransferase enzyme encoded by the ABO gene. Variants in the ABO gene can produce a weak ABO phenotype. In this study, we identify a novel ABO*BW allele and investigate the underlying mechanism leading to the Bweak phenotype. The ABO phenotype and genotype of the sample were determined using serological and direct DNA sequencing methods. We assessed the impact of the novel variant by three-dimensional modelling to predict protein stability changes (ΔΔG), and carried out an in vitro expression assay. The total glycosyltransferase transfer capacity in the supernatant of transfected cells was also examined. Serological analysis confirmed the Bweak phenotype in the subject, and gene sequencing identified a novel variant c.761C>T (p.A254V) on the ABO*B.01 allele, resulting in a BW-var/O.01.02 genotype. In silico analysis suggested that the p.A254V variant on the B allele may reduce the stability of glycosyltransferase B (GTB), as indicated by the ΔΔG values. In vitro expression studies showed that the variant p.A254V impaired H to B antigen conversion, although it did not affect the expression of GTB. We identified a novel BW allele and demonstrated that the variant c.761C>T (p.A254V) can cause the Bweak phenotype by reducing the stability of GTB.

Establishment of Biotesting System to Study Features of Innovative Multifunctional Biotextile

Abstract An established biotesting system designed to discover specific features of innovative multifunctional biotextile, encompassing integrated silica dioxide and succinite (Baltic amber) particles, is founded on reproducible, reliable, and relatively fast methods. The main idea starting this study was to create a system of test methods devoted to identification of specific features of biotextile materials designed to preserve living organisms from adverse environmental factors like enhanced electromagnetic radiation of different frequencies, without use of vivarium animals. Cultures of the freshwater macrophyte duckweed (Lemna minor) line Sta2 and fruit flies (Drosophila melanogaster) were chosen as model systems suitable for the study of the influence of electromagnetic field (EMF) radiation. The experiments showed changes of phenotypic features and growth parameters of test objects, as well as induction of point mutations of DNA (for example, insertions or deletions in chloroplast DNA and nucleotide substitutions in nuclear genes). The responses of test organisms induced by EMF were studied using microscopy, flow cytometry, and DNA sequencing methods. On the cell level, a new fast flow cytometry method for biotextile testing was developed: immature gametic (pollen) cell cultures were used as a highly sensitive model system (plant gametic cell response is comparable to human neutrophil response) to study the influence of EMF radiation. The method was based on the measurement of differences of fluorescence intensity between group of cells experimentally affected by EMF radiation and non-affected cells.

Archival single-cell genomics reveals persistent subclones during DCIS progression

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

Chaotic-based color image encryption using a hybrid method of reversible cellular automata and DNA sequences

Chaotic-based color image encryption using a hybrid method of reversible cellular automata and DNA sequences

Machine learning-based approaches for cancer prediction using microbiome data

Emerging evidence of the relationship between the microbiome composition and the development of numerous diseases, including cancer, has led to an increasing interest in the study of the human microbiome. Technological breakthroughs regarding DNA sequencing methods propelled microbiome studies with a large number of samples, which called for the necessity of more sophisticated data-analytical tools to analyze this complex relationship. The aim of this work was to develop a machine learning-based approach to distinguish the type of cancer based on the analysis of the tissue-specific microbial information, assessing the human microbiome as valuable predictive information for cancer identification. For this purpose, Random Forest algorithms were trained for the classification of five types of cancer—head and neck, esophageal, stomach, colon, and rectum cancers—with samples provided by The Cancer Microbiome Atlas database. One versus all and multi-class classification studies were conducted to evaluate the discriminative capability of the microbial data across increasing levels of cancer site specificity, with results showing a progressive rise in difficulty for accurate sample classification. Random Forest models achieved promising performances when predicting head and neck, stomach, and colon cancer cases, with the latter returning accuracy scores above 90% across the different studies conducted. However, there was also an increased difficulty when discriminating esophageal and rectum cancers, failing to differentiate with adequate results rectum from colon cancer cases, and esophageal from head and neck and stomach cancers. These results point to the fact that anatomically adjacent cancers can be more complex to identify due to microbial similarities. Despite the limitations, microbiome data analysis using machine learning may advance novel strategies to improve cancer detection and prevention, and decrease disease burden.

Manipulative-based Activity Using Pop Beads for Demonstration of Sanger Sequencing.

Sanger sequencing, also known as dideoxy sequencing, is a widely used method for DNA sequencing, particularly for cloned plasmids and clinical samples. This technique requires a combination of essential biochemistry skills, such as a chain-termination reaction, gel electrophoresis, and fluorescence detection. Unfortunately, there is a lack of activities that replicate the Sanger sequencing process for students to learn and practice these skills. To address this issue, a manipulative-based Sanger sequencing activity was developed that incorporates colorful pop beads to demonstrate a chain-termination reaction, separation of products, and fluorescence detection. The beads represent deoxynucleotides and dideoxynucleotides, allowing for a visual representation of DNA fragment generation. This kinesthetic learning activity offers a high visual impact for students, aiding in their understanding of the Sanger sequencing process, and can also be used to illustrate polymerase chain reaction (PCR)-based techniques.

Nanopore Sequencing of Short dsDNA after Elongation by Combination of Ligation and PEAR

Abstract Nanopore sequencing technology, as a third-generation method for DNA sequencing at the single-molecule level, has attracted much attention and developed quickly due to the advantages of low cost and ultra-long read. However, nanopore sequencing of short DNAs (<500 bp) is not suitable due to the unfavorable cost performance and complicated data analysis. Here, we describe a novel method for nanopore sequencing using short dsDNA elongation by ligation and PEAR (SELP). Before sequencing, short PCR products are subjected to intermolecular ligation and subsequent elongation using PEAR (Polymerase-Endonuclease Amplification Reaction). The obtained long concatemers (thousands of base pairs) of repetitive DNA sequences are ideal samples for nanopore sequencing. The sequencing results demonstrate that short dsDNA can be elongated by more than 20 fold, and precise sequence analysis can be obtained through a single read. Accordingly, SELP-Seq can be used for simultaneous sequencing of multiple (even thousands of) short dsDNAs. Obviously, our approach can greatly expand the applications of nanopore sequencing, such as SNP analysis and high-throughput DNA detection.

Screening for causative mutations in ovine BMPR1B and BMP15 genes and their homologous fragments in human.

The BMPR1B and BMP15 genes are well known for their considerable associations with prolificacy in sheep. These genes may also affect fertility or prolificacy in other species, including human. This study was conducted to investigate possible causative mutations in BMPR1B and BMP15 genes in human and an indigenous breed of sheep. Blood samples were collected from 83 singleton- and prolific Mehraban ewes and 81 infertile, singleton- and twin-bearing women. A 190-bp fragment, containing the FecB mutation in ovine BMPR1B, a 380-bp fragment in ovine BMP15 gene and their homologous fragments in human were amplified and then investigated by single-stranded conformation polymorphism and DNA sequencing methods. The FecB mutation of BMPR1B (g.159A>G) was detected in the sheep population, but no polymorphic loci were found in the homologous fragment in studied human samples. The studied fragments of BMP15 were monomorphic in both sheep and human samples. A total of nine and 69 point-differences in the studied fragments of BMPR1B and BMP15 genes were detected between the species, respectively. In sheep, the G allele of BMPR1B had a positive effect on litter size (p<0.05), whereby all AG or GG ewes were prolific. The FecB mutation for the first time was detected in Mehraban sheep and therefore could be considered for marker-assisted selection in this breed. The studied fragments of BMPR1B and BMP15 genes are not responsible for reproduction variation in human. More studies on other genes, associated with fertility in human, are necessary in the future.

Guidelines for Microbiome Studies in Renal Physiology.

Gut microbiome research has increased dramatically in the last decade, including in renal health and disease. The field is moving from experiments showing mere association to causation using both forward and reverse microbiome approaches, leveraging tools such as germ-free animals, treatment with antibiotics, and faecal microbiota transplantations. However, we are still seeing a gap between discovery and translation that needs to be addressed, so that patients can benefit from microbiome-based therapies. In this guideline paper, we discuss the key considerations that affect the gut microbiome of animal and clinical studies assessing renal function, many which are often overlooked, resulting in false-positive results. For animal studies, these include suppliers, acclimatisation, baseline microbiota and its normalisation, littermates and cohort/cage effects, diet, sex differences, age, circadian differences, antibiotics and sweeteners, and models used. Clinical studies have some unique considerations, which include sampling, gut transit time, dietary records, medication, and renal phenotypes. We provide best practice guidance on sampling, storage, DNA extraction, and methods for microbial DNA sequencing (both 16S rRNA and shotgun metagenome). Finally, we discuss follow-up analyses, including tools available, metrics, and their interpretation, and the key challenges ahead in the microbiome field. By standardising study designs, methods and reporting, we will accelerate the findings from discovery to translation and result in new microbiome-based therapies that may improve renal health.

Performance Analysis of Parallel Implementations of Global Sequence Alignment Methods for DNA Sequencing

Performance Analysis of Parallel Implementations of Global Sequence Alignment Methods for DNA Sequencing

P-724 The choice of genetic service provider can have significant implications for the outcome of IVF cycles using preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Does the choice of PGT-A provider have any impact on clinical results, such as rates of aneuploidy, pregnancy and miscarriage? Summary answer The laboratory providing PGT can have a significant impact on various important clinical outcomes. PGT-A providers are not equivalent and should be chosen with care. What is known already A growing number of IVF cycles include PGT-A to assist in the identification of euploid embryos for transfer to the uterus. All modern PGT laboratories utilise a method called next generation sequencing (NGS) to predict the copy number of each chromosome in embryo biopsy samples. Given this technical convergence, it is often supposed that genetic results will be similar regardless which PGT laboratory is used, and consequently a clinic choosing a genetics provider need only consider convenience, quality of support and price. Here we consider whether the choice of PGT-A provider might have more profound affects, potentially impacting clinical results. Study design, size, duration A large network of IVF clinics switched from PGT-A provider ‘A’ to provider ‘B’. The final 6 months of clinical data using provider A was compared to 6 months of data after the switch to B. There were no changes in any aspect of patient population, treatment protocols, or embryological practice occurred between the two time periods evaluated. Genetic results (aneuploidy/euploidy rates) and clinical outcomes (implantation, miscarriage, ongoing pregnancy/birth) were compared to identify any differences. Participants/materials, setting, methods Within the two time periods considered, 9,091 embryos underwent PGT-A with provider A and 10,281 using B. The average female age was 39.0 and 39.3, respectively. Embryos were biopsied at the blastocyst stage and the cells were shipped to the genetics laboratories. Provider A was in the same country as the IVF clinics, while provider B was located overseas. Both PGT-A laboratories used NGS, but employed different methods for DNA amplification, sequencing and data analysis. Main results and the role of chance A higher proportion of embryos were classified euploid by provider B in comparison to A (49.3% versus 39.8%; p &amp;lt; 0.0001). Differences in reported aneuploidy were particularly striking for younger patients (&amp;lt;38 years; 67.5% euploid according to B versus 52.5% reported by A; p &amp;lt; 0.0001). For patients over 37 years, the lower aneuploidy frequency reported by B resulted in fewer cycles with all embryos classified ‘abnormal’ and more cycles with a transfer (52.2% of 2115 cycles in this age group had a transfer, compared to 48.6% of 1915 cycles for A; p = 0.02). Having more embryos classified euploid also benefitted younger patients, providing opportunities to combine morphological selection with genetic evaluation. Improved selection may explain a higher ongoing pregnancy rate observed for patients &amp;lt;38 years (63.2% ongoing after the first embryo transfer for B versus 55.3% for A). Differing miscarriage rates were also observed (18.1% when using A versus 15.1% for B; p = 0.047). We speculate that higher rates of euploidy and pregnancy when using provider B might be a consequence of viable embryos being excluded due to incorrect classification as ‘abnormal’ by A. Additionally, failure to detect some aneuploidies, leading to transfer of abnormal embryos, might explain the higher miscarriage rate associated with A. Limitations, reasons for caution There were no detectable differences in the composition of patient populations receiving PGT-A from the two providers, and no apparent changes to protocols used during the two time periods assessed. Nevertheless, it must be acknowledged that the conclusions are based on a retrospective review of data, not a controlled study. Wider implications of the findings PGT-A and NGS are umbrella terms encompassing multiple methods with widely varying levels of validation and accuracy. Our results suggest that the choice of PGT-A provider has important implications for clinical results, affecting the number of transferrable embryos, pregnancy, and miscarriage rates. The observed differences likely reflect differing PGT-A accuracies. Trial registration number Not applicable

O-060 Single-cell DNA sequencing reveals that current bulk DNA sequencing methods lead to an underestimation of chromosomal mosaicism in human blastocysts

Abstract Study question What is the incidence of chromosomal mosaicism in trophectoderm and inner cell mass from good quality human blastocysts after NGS-based single-cell molecular karyotyping? Summary answer Single-cell analysis showed that almost all human blastocysts are mosaic, involving low level mosaicism and/or abnormal cells resulting from reciprocal error events. What is known already Chromosome segregation in early human embryos is considered to be error prone. While meiotic errors affect all cells within embryos, mitotic errors lead to chromosomal mosaicism, the presence of cytogenetically different cells within an embryo. The reported incidence of mosaicism in blastocysts after bulk DNA sequencing of multicellular trophectoderm (TE) biopsies performed for preimplantation genetic testing for aneuploidy (PGT-A) varies between 2-19%. However, bulk DNA analysis can only assess net average chromosome gains or losses with a detection limit of 20-30%, and it makes distinguishing true mosaicism from technical artefact problematic. Study design, size, duration Observational study in human good quality surplus embryos which were donated for research purposes (CCMO, NL82597.000.22). The embryos were thawed and cultured until the blastocyst stage. From the blastocysts with at least a morphology grade 3BB, the ICM was dissociated from the TE. From 55 embryos, both samples were successfully disaggregated into single cells and manually placed in 384 well plates. For the cytogenetic analysis, a validated method for single-cell molecular karyotyping was used (scKaryo-seq). Participants/materials, setting, methods Embryos with at least two cytogenetically different cells were considered mosaic. To investigate if bulk DNA sequencing would have detected this mosaicism, we performed an in-silico reanalysis on our single-cell data. For each embryo, TE cells with the same mitotic abnormality were quantified. Next, we determined the number of mosaic embryos in which these abnormalities were present in at least 20% of TE cells, as a cut-off for what PGT-A methods are expected to detect. Main results and the role of chance On average 42% of the total number of cells that could be isolated per embryo were successfully karyotyped, giving a total of 1057 karyotyped cells (522 normal, 535 abnormal) from 55 embryos. Six embryos (11%) were normal, four (7%) were uniformly abnormal and 45 (82%) were mosaic. From these, 14 (26%) embryos were aneuploid mosaic with cytogenetically different abnormal cells and 31 (56%) were diploid-aneuploid mosaic with normal and abnormal cells. Here cytogenetically different abnormal cells were also frequently observed, with products of reciprocal events detected in 20 embryos. The in-silico reanalysis was performed on 38 embryos with a mosaic TE. It predicted that only 11% of the mitotic abnormalities observed in TE cells through scKaryo-seq would have been detected by bulk DNA sequencing. In 19 out of 38 embryos at least one mitotic abnormality affected more than 20% of the TE cells, meaning that only 50% of the embryos would have been recognized as mosaic with bulk DNA analysis. Bulk DNA sequencing methods used for PGT-A lead to an underestimation of mosaicism, since low-level mosaicism that affects a few cells within an embryo and products of reciprocal events without a net gain or loss remain undetected. Limitations, reasons for caution Isolation and cytogenetic analysis of viable single cells at the blastocyst stage is technically challenging and only 42% of the cells per embryo could be successfully karyotyped. Furthermore, it is unknown to what extent in vitro culture conditions and the freezing-thawing process influenced our findings. Wider implications of the findings These single cell observations support the notion that mosaicism is a common biological phenomenon in human blastocysts. Any selective mechanism eliminating embryos or cells with chromosomal abnormalities is still not fully active at this stage. The underestimation of mosaicism by bulk DNA analysis has potential implications for current PGT-A practices Trial registration number not applicable

HbAdrian (α1:c.251del, p.Leu84Argfs*19)—A Novel Pathogenic Variant in the α1-Globin Gene Associated with Microcytosis from the North of Iran

Background: Alpha thalassemia is one of the most common human genetic abnormalities. More than 400 different variations of the α-globin protein have been introduced, most of which are not associated with noticeable clinical manifestations. The identification of all variants of Hb in different regions helps in acquiring comprehensive knowledge concerning thalassemia disease, and it can be used in preventive programs as well as prenatal diagnosis (PND). Aims: In the present study, we describe a new α1 gene mutation that leads to a frameshift after codon 83. Methods: As a plan for a national screening program of thalassemia, routine cell blood count (CBC) and Hb capillary electrophoresis tests were applied. After taking written informed consent, genomic DNA was extracted, and, for identifying common Mediterranean α-Globin gene deletion, multiplex Gap-PCR was performed; for detecting other mutations on α- and β-Globin genes, a DNA sequencing method was used. Results: The results of CBC and capillary electrophoresis tests showed microcytosis in a female subject. The sequencing of the α-Globin gene showed that the case is heterozygote for a single-nucleotide deletion at codon 83 of the α1-Globin Gene. We named this mutation Hb Adrian (α1: c.251–T), which is a novel mutation. The mentioned mutation was also detected in the subject’s mother. Conclusions: The introduced mutation (Hb Adrian) leads to a frameshift change that produces a protein with 100 amino acids, which in comparison to a normal α-chain is shorter, and its amino acids are altered after codon 83. This hemoglobin is undetectable via the use of electrophoresis. Although no major hematological abnormalities were observed in the carriers, Hb Adrian should be considered in screening programs to help prevent Hb H disease in high-risk couples.

Wood-decay fungi associated with decaying of coarse woody debris in Hail Region, North of Saudi Arabia.

The present study was conducted in order to describe the assemblage of wood decay fungi and mycomycetes associated with the Hail region in the North of Saudi Arabia by using incubation chambers. There was a collection of coarse woody debris from the study area, AL baithe, which was brought to the laboratory and placed inside a series of incubation chambers in which water was added. Over a period of several weeks, fruiting bodies appeared in these chambers which were morphologically analysed via AmScope stereomicroscope and molecular identification was carried out via ITS-1F and ITS-4R DNA sequencing methods. The obtained sequences after successful identification via BLAST analysis were submitted to the Genebank NCBI database. During the course of the study, a total of 20 species of different 14 families of wood-decay fungi were identified of which mycelium has been generated in incubation chambers. Among the identified wood-decay fungi, Phanerochaetaceae (03 species), Bionectriaceae (02 species), Hypocreaceae (02 species) and Chaetomiaceae (02 species) being the most frequent. Hence, the present study was the first one to report on the diversity of wood-decay fungi of Saudi Arabia and was successful in using incubation chambers to describe the specimens of wood-decay fungi associated with coarse wood debris.