DNA Segments Research Articles

Mosaic Chromosomal Alterations and Monoclonal B-Cell Lymphocytosis: Relationship and Risk of Hematologic Malignancies

Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor condition to chronic lymphocytic leukemia (CLL) characterized by the presence of circulating monoclonal B-cell populations in the peripheral blood of otherwise healthy individuals. MBL increases risk of several adverse clinical outcomes, including development of hematological malignancies. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis which affect large segments of DNA and include gains, losses, and copy number neutral loss of heterozygosity events. mCAs can be detected from SNP-array genotyping. Prior work has suggested mCAs as a potent risk factor for hematological malignancies. However, the nature of the relationship between mCAs and MBL is unknown. We sought to determine 1) the association between mCAs and MBL and 2) their combined effect on risk of hematological malignancies, in a large, prospectively collected, and well-annotated cohort of individuals with known MBL, mCA, and clinical outcomes. Methods: We examined the prevalence of mCA in a cohort of individuals from the Mayo Clinic Biobank who were screened for MBL using a sensitive eight-color flow cytometry assay. We classified MBL by the size of the clone relative to total B-cells, with high count (HC) defined as those individuals who had a percent clonal B-cell count ≥85% out of total B-cell count and low-count (LC) otherwise. Autosomal mCAs were detected from whole blood DNA collected at the time of MBL screening using a SNP-array-based assay. The MoChA pipeline was used to identify mCA events. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). To examine incident hematological cancers, we identified and validated all hematological cancers through medical record abstraction using the International Statistical Classification of Diseases codes. Cox regression was used to calculate hazard ratios (HR) and 95% CIs, stratified by MBL status or autosomal mCA status. All models were adjusted for age and sex. Results: We evaluated the prevalence of autosomal mCA in 3,764 individuals (792 individuals with MBL of whom 64 had HC-MBL and 2,972 without MBL). The prevalence of autosomal mCA was 7.7% ( n=288) with a median mCA cell fraction of 0.09 (range 0.01-1). Among those with and without MBL overall, the prevalence of mCA was 15.5% ( n=123) and 5.6% ( n=165), respectively (Figure 1). Compared to those without an autosomal mCA, individuals with an autosomal mCA were 2.4-fold more likely to have MBL (95% CI 1.9-3.2; P =2.0x10 -11; Figure 1). CLL-associated mCA (i.e., those commonly altered in CLL including trisomy 12 and deletion of 6q, 11q, 13q, and 17p) were present in 42 individuals, of whom 38 (90.5%) had MBL overall and 30 (71.4%) individuals had HC-MBL. Compared to those without a CLL-associated mCA, individuals with a CLL-associated mCA were 539.39-fold more likely to have HC-MBL (95% CI 190.3-1975.9; P <2.0x10 -16) and 6.7-fold more likely to have LC-MBL (95% CI 2.0-26.5; P =0.0031; Figure 1). Next, we evaluated the relationship between MBL and mCA with risk of developing a hematologic malignancy in 3,009 individuals with follow-up data (median 10 years). Forty-one individuals developed an incident hematological malignancy (68.3% lymphoid). When we evaluated the effect of autosomal mCA stratified by MBL status we observed elevated but non-significant risk of hematological malignancies (Figure 2). When we evaluated the effect of MBL stratified by autosomal mCA status we observed elevated and significant increased risk of developing a hematological malignancy (Figure 2). Conclusions: We found that the presence of an autosomal mCA is highly associated with having MBL. In particular, >90% of CLL-associated mCA carriers had MBL with ~7- and ~540-fold increased association for LC- and HC-MBL, respectively. These results suggest that almost all individuals in large biobanks with CLL-associated mCAs have undiagnosed MBL. However, not all individuals with MBL have an mCA. When evaluating risk of hematological malignancies, we found autosomal mCAs had >2-fold elevated but non-significant effects when stratified by MBL status while MBL remained a significant risk factor regardless of autosomal mCA status. Collectively these results support that MBL remains an important risk factor and suggests that mCAs hold potential to identify a subgroup of individuals at greater risk of developing a hematological malignancy.

A Novel Predictor for the Analysis and Prediction of Enhancers and Their Strength via Multi-View Features and Deep Forest

Enhancers are short DNA segments (50–1500 bp) that effectively activate gene transcription when transcription factors (TFs) are present. There is a correlation between the genetic differences in enhancers and numerous human disorders including cancer and inflammatory bowel disease. In computational biology, the accurate categorization of enhancers can yield important information for drug discovery and development. High-throughput experimental approaches are thought to be vital tools for researching enhancers’ key characteristics; however, because these techniques require a lot of labor and time, it might be difficult for researchers to forecast enhancers and their powers. Therefore, computational techniques are considered an alternate strategy for handling this issue. Based on the types of algorithms that have been used to construct predictors, the current methodologies can be divided into three primary categories: ensemble-based methods, deep learning-based approaches, and traditional ML-based techniques. In this study, we developed a novel two-layer deep forest-based predictor for accurate enhancer and strength prediction, namely, NEPERS. Enhancers and non-enhancers are divided at the first level by NEPERS, whereas strong and weak enhancers are divided at the second level. To evaluate the effectiveness of feature fusion, block-wise deep forest and other algorithms were combined with multi-view features such as PSTNPss, PSTNPdss, CKSNAP, and NCP via 10-fold cross-validation and independent testing. Our proposed technique performs better than competing models across all parameters, with an ACC of 0.876, Sen of 0.864, Spe of 0.888, MCC of 0.753, and AUC of 0.940 for layer 1 and an ACC of 0.959, Sen of 0.960, Spe of 0.958, MCC of 0.918, and AUC of 0.990 for layer 2, respectively, for the benchmark dataset. Similarly, for the independent test, the ACC, Sen, Spe, MCC, and AUC were 0.863, 0.865, 0.860, 0.725, and 0.948 for layer 1 and 0.890, 0.940, 0.840, 0.784, and 0.951 for layer 2, respectively. This study provides conclusive insights for the accurate and effective detection and characterization of enhancers and their strengths.

Unexpectedly high prevalence of structural genomic variations in the Long QT Syndrome: evidence for broader clinical uptake

Abstract Background Long QT syndrome (LQTS) is an inherited channelopathy predisposing young and otherwise healthy individuals to ventricular arrhythmias causing syncope or sudden cardiac death. Point mutations in the KCNQ1, KCNH2, and SCN5A genes are the most common causes of LQTS, and up to 75% of patients can be genotyped by standard DNA sequencing. However, standard genetic testing fails to detect structural genomic rearrangements - including large deletions or duplications of DNA segments - known as copy number variants (CNVs). While previous studies suggested that CNVs account for less than 5% of genotype-negative cases(1-2), no recent data have been reported. Purpose The study aimed to determine the prevalence and spectrum of CNVs in LQTS susceptibility genes in unrelated patients who tested negative for point mutations in a panel of 16 genes associated with LQTS. Methods Next-generation sequencing (NGS) was used to screen CNVs in unrelated, clinically diagnosed LQTS patients without point mutations in 16 genes associated with LQTS. Loss of heterozygosity in LQTS-associated genes was used to assess the presence of CNVs using standard commercial software. The multiplex ligation-dependent probe amplification (MLPA) assay was used to confirm the CNVs identified by NGS. Results The cohort included 48 patients (63% women; mean age 31±19 years, range 3 years to 68 years) with definitive clinical diagnosis of LQTS (mean QTc 490±33 ms) according to the 2022 ESC guidelines(3). Overall, 7/48 patients (15%, 95% CI 6-28%) had CNVs. Six of the seven CNVs involved KCNQ1 in the heterozygous state, including three large deletions (exons 3-6, exons 12-15, and exon 16) and three duplications (exon 2, exons 3-6, and exons 3-15), whereas the remaining patient had a complete KCNH2 deletion. MLPA confirmed all CNVs identified by NGS. Of note, no CNVs were identified in LQTS-related genes whose pathogenicity has been questioned. Conclusions Our study highlights the unexpectedly high prevalence of structural genomic rearrangements in the KCNQ1 and KCNH2 genes in LQTS patients. Routine screening for copy number variants in LQTS patients is critical for accurate diagnosis, risk stratification, and personalized treatment strategies.Figure 1

Most large structural variants in cancer genomes can be detected without long reads

Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed ‘loose ends’—local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.

Ecological monitoring using Collembola metabarcoding with extremely low bycatch amplification

Collembola are used widely to monitor soil health and functional parameters. Recent developments in high throughput sequencing (especially metabarcoding) have substantially increased their potential for these ends. Collembola are especially amenable to metabarcoding because of their small size, high abundance, and ubiquity in most habitat types. However, most Collembola sampling protocols collect a substantial and highly varied bycatch that can be a considerable impediment to metabarcoding, especially because of data lost to non-target species. We designed a primer set amplifying the D2 expansion segment of ribosomal DNA that is highly conserved across Collembola and successfully excludes from amplification nearly all other invertebrate taxa. We tested the diagnostic power of the primer set by clearly distinguishing Collembola communities between forest sites with differing habitat qualities in São Paulo State, Brazil. The oligos amplified all Collembola orders previously encountered in the sampling locations, with no non-target amplification, and also excluded the closely related Protura and Diplura. Alpha diversity (OTU count) and phylogenetic diversity was significantly higher in high quality habitats. Moreover, the beta diversity indices successfully differentiated high and low-quality habitats. This new addition to the biomonitoring toolbox greatly increases the accessibility of Collembola metabarcoding for various types of habitat assessments.

C-176 loaded Ce DNase nanoparticles synergistically inhibit the cGAS-STING pathway for ischemic stroke treatment.

The neuroinflammatory responses following ischemic stroke cause irreversible nerve cell death. Cell free-double strand DNA (dsDNA) segments from ischemic tissue debris are engulfed by microglia and sensed by their cyclic GMP-AMP synthase (cGAS), which triggers robust activation of the innate immune stimulator of interferon genes (STING) pathway and initiate the chronic inflammatory cascade. The decomposition of immunogenic dsDNA and inhibition of the innate immune STING are synergistic immunologic targets for ameliorating neuroinflammation. To combine the anti-inflammatory strategies of STING inhibition and dsDNA elimination, we constructed a DNase-mimetic artificial enzyme loaded with C-176. Nanoparticles are self-assembled by amphiphilic copolymers (P[CL35-b-(OEGMA20.7-co-NTAMA14.3)]), C-176, and Ce4+ which is coordinated with nitrilotriacetic acid (NTA) group to form corresponding catalytic structures. Our work developed a new nano-drug that balances the cGAS-STING axis to enhance the therapeutic impact of stroke by combining the DNase-memetic Ce4+ enzyme and STING inhibitor synergistically. In conclusion, it is a novel approach to modulating central nervus system (CNS) inflammatory signaling pathways and improving stroke prognosis.

Characterization of the marsupial endogenous retrovirus walb with a focus on satellite DNA formation

The walbRep megasatellite DNA found in the red-necked wallaby was formed from the walb endogenous retrovirus. Our previous PCR experiments suggested the presence of walb and absence of walbRep in the genome of the tammar wallaby, which diverged from the red-necked wallaby 2-3 Mya. The results failed to exclude the possibility that certain walbRep sequences might have remained undetected owing to variation in the primer-annealing regions; therefore, the aforementioned suggestion was not confirmed. To obtain conclusive evidence, we analyzed the structure of walb sequences drawn from the tammar wallaby genome database recently updated to a chromosome-level assembly. All walb copies existed as separate DNA segments, not constituting tandem repeats. We concluded that walbRep was formed in the red-necked wallaby lineage after its divergence from the tammar wallaby. We also confirm the presence of a walb copy with an anomalistic, complex structure and propose a simple model for its generation mechanism.

The association of positive sepsis PCRs tests with patient mortality, length of stay, and antibiotic management in a medical intensive care unit

Background: Polymerase chain reaction (PCR) testing amplifies a specific DNA segment through heat cycling to identify pathogens for the diagnosis of infection. This testing allows for rapid detection of pathogens, before conventional blood culture results become available. The high mortality associated with severe sepsis and septic shock stresses the importance of early diagnosis and initiation of early antibiotic therapy. This study analyzed the association of the sepsis PCR results and antibiotic management, length of stay, and mortality outcomes in a medical intensive care unit (MICU).
 Methods: This study is a retrospective, cross-sectional study on patients diagnosed with severe sepsis and septic shock based on ICD-10 codes, aged 18 years and older, admitted to the MICU at University Medical Center, Lubbock, Texas, between December 2016 to December 2020.
 Results: Clinical information from 268 patients with the diagnosis of sepsis or septic shock was collected and analyzed. The mean age was 60.9 ± 15.6 years with a predominance of men (144, 53.7%) and Caucasian race (193,73.4%). A total of 101 patients (37.6%) had positive PCR results; overall, PCR test results had a diagnostic sensitivity of 91.2 %. The concordance between positive blood culture and positive PCR was 93.3 % (p <.0001). There was a significant correlation between PCR positivity and increased serum lactate levels (p=0.03), changes in antibiotics (P<0.00010), and increased mortality rate (p=0.04). There was no significant correlation between PCR positivity and length of stay (p=0.84).
 Conclusion: In this study, PCR testing was an accurate tool for early identification of bacterial pathogens. A positive PCR was associated with higher serum lactate levels, higher mortality rates, and an increased frequency of antibiotic changes but was not associated with shorter ICU length of stay.
 Key words: Sepsis, septic shock, PCR testing, outcomes

Advantages and Challenges of CRISPR-Cas9 Applications in Animal Modeling: A Concise Review CRISPR-Cas9 Applications in Animal Modeling

CRISPR is an extraordinarily powerful technique regulating any target gene across the genome with promising therapy intentions. CRISPR-Cas9 is a convenient tool for gene manipulation. Notwithstanding this, the broad consequence of human gene editing, particularly germinal genes, cannot be predicted. Firstly, once edited, the genes would be part of the human population for successive generations and may be impossible to remove from humanity; secondly, success is not guaranteed; thirdly, the fidelity of editing, as it could affect unrelated genes or unspecified segments of DNA; and last but not least, its influence on gene interaction, network, and signaling pathways could be difficult to be predicted. CRISPR-Cas9 mostly includes precise genome editing, rapidity and cost-effectiveness, creation of disease models, study of gene function, applications in gene therapy and translation research and wide diversity for species. The technique also ignited the moral and ethical concerns of scientific community. Ethics and safety approval for gene modification in human cells is required by the National Institutes of Health (NIH). The NIH does not currently fund studies of CRISPR in human embryos and opposes the CRISPR utilization in germline cells because these alterations would be permanent and heritable. The technology has promised with the most profound implications for cancer therapy. Recent advances in CRISPR-based technology is redefining how cancer is studied and potentially improves anti-cancer therapies. One way to improve the technology is to use machine-learning approaches to comprehending CRISPR errors and predicting more specific edits and repairing outcomes.

Magnetically actuated microfluidic chip combined with a G-quadruplex DNAzyme-based fluorescent/colorimetric sensor for the dual-mode detection of ochratoxin A in wheat

In this work, a portable fluorescent/colorimetric sensor based on G-quadruplex DNAzyme was constructed to achieve rapid and dual-mode detection of ochratoxin A (OTA) in wheat. OTA aptamers coupled with magnetic beads (MBs) can self-assemble with two segments of DNA and hemin to form a G-quadruplex DNAzyme structure that can catalyze the oxidation of Amplex Red (ADHP) with H2O2, making the solution red and producing strong fluorescence in solution. However, in the presence of OTA, the structure of the G-quadruplex DNAzyme was damaged, resulting in reduced catalytic activity. According to the principle of detection, a magnet-controlled chip integrating the reaction, washing, and detection was designed in this study. Shuttling the MB-DNAzyme probes onto a magnetically controlled chip considerably reduced the background signal and improved the detection efficiency and sensitivity. In addition, a portable fluorescence and colorimetric detection platform was built for on-site OTA detection.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

SNN6mA: Improved DNA N6-methyladenine site prediction using Siamese network-based feature embedding

DNA N6-methyladenine (6mA) is one of the most common and abundant modifications, which plays essential roles in various biological processes and cellular functions. Therefore, the accurate identification of DNA 6mA sites is of great importance for a better understanding of its regulatory mechanisms and biological functions. Although significant progress has been made, there still has room for further improvement in 6mA site prediction in DNA sequences. In this study, we report a smart but accurate 6mA predictor, termed as SNN6mA, using Siamese network. To be specific, DNA segments are firstly encoded into feature vectors using the one-hot encoding scheme; then, these original feature vectors are mapped to a low-dimensional embedding space derived from Siamese network to capture more discriminative features; finally, the obtained low-dimensional features are fed to a fully connected neural network to perform final prediction. Stringent benchmarking tests on the datasets of two species demonstrated that the proposed SNN6mA is superior to the state-of-the-art 6mA predictors. Detailed data analyses show that the major advantage of SNN6mA lies in the utilization of Siamese network, which can map the original features into a low-dimensional embedding space with more discriminative capability. In summary, the proposed SNN6mA is the first attempt to use Siamese network for 6mA site prediction and could be easily extended to predict other types of modifications. The codes and datasets used in the study are freely available at https://github.com/YuXuan-Glasgow/SNN6mA for academic use.

High Mitochondrial Haplotype Diversity Found in Three Pre-Hispanic Groups from Colombia.

The analysis of mitochondrial DNA (mtDNA) hypervariable region (HVR) sequence data from ancient human remains provides valuable insights into the genetic structure and population dynamics of ancient populations. mtDNA is particularly useful in studying ancient populations, because it is maternally inherited and has a higher mutation rate compared to nuclear DNA. To determine the genetic structure of three Colombian pre-Hispanic populations and compare them with current populations, we determined the haplotypes from human bone remains by sequencing several mitochondrial DNA segments. A wide variety of mitochondrial polymorphisms were obtained from 33 samples. Our results support a high population heterogeneity among pre-Hispanic populations in Colombia.

Molecular and Systemic Epigenetic Inheritance: Integrating Development, Genetics, and Evolution

Currently, a central problem for theoretical biology is the integration of development with genetics and evolutionary theory. Through the late 20th century, biologists held that animals resemble their ancestors strictly because of the transgenerational transmission of DNA. This view effectively wrote development out of evolutionary biology. However, many molecular and developmental biologists now understand that phenotypes – anatomical, physiological, and behavioral traits – are not determined by genes (i.e., DNA segments) alone; instead, they emerge epigenetically from developmental processes involving co-acting genetic factors, environmental factors, molecular epigenetic factors, and other non-genetic factors within organisms’ bodies. This insight forces a rethinking of biological inheritance. Perspectives focusing on the dynamics of developmental systems offer a compelling alternative way to think about inheritance, providing a powerful substitute to the reductionistic framework that attributes phenotypic outcomes to genetic instructions set in advance of developmental processes. Rethinking genetics, epigenetics, and inheritance by focusing on the dynamics of developmental systems helps highlight the bidirectional effects of evolutionary and developmental processes on one another, yielding a more integrated understanding of development, inheritance, and evolution. Simultaneously, this approach encourages rejection of genetic determinism, a simplistic perspective that continues to appear in psychological writing, despite its biological implausibility.

383. DEVELOPMENT OF PERSONALIZED ‘LIQUID BIOPSY’ FOR ESOPHAGEAL ADENOCARCINOMA FROM MATCHED ORGANOID CULTURE

Abstract Background Recurrent mutations are not a hallmark of esophageal adenocarcinoma (EAC). This challenges the identification of circulating tumor DNA in the plasma and can limit the development of liquid biopsy strategies. We have cultured patient-derived EAC organoids and hypothesize that these can be used as a roadmap for the identification of circulating tumor DNA in the corresponding patient’s blood. Methods EAC tumor tissue from patients was processed to generate patient-derived organoids (PDOs) in Matrigel domes. Established PDOs were then scaled-up in suspension culture. To isolate mononucleosomes (147 bp), chromatin from these cells was extracted and digested with micrococcal nuclease (MNase). Right size selection was used to remove DNA larger than the targeted 147 bp. Next Generation Sequencing will be performed for nucleosome mapping and to generate a personlized SNV map. Using the SNV map, a personalized PCR panel will be developed to detect circulating tumor somatic variants in patient plasma cfDNA. Results DNA from three different PDOs were collected and MNase digested. MNase concentration and digestion time were optimized for each sample to avoid over digestion. MNase digestion resulted in mononucleosomes at approximately 147 bp for all three samples, as well as large DNA segments between 300 to 8000 bp. Right size selection resulted in isolation of 34 to 48 ng of the mononucleosome peak. These results indicate that MNase digestion was successful in generating nucleosomes of the desired size range and the selected nucleosomes were of high quality, as confirmed by the Bioanalyzer. Samples will be subjected to next-generation sequencing. Conclusion This study will determine nucleosome SNVs maps in circulating cell-free DNA from EAC patients to allow for generation of optimized targeted PCR panels, prediction of recurrence based on specific variants unique to each patient, and development of more precise drug screening. These advantages have the potential to fill a void in early cancer detection and the prediction of cancer recurrence, leading to improved prognoses for individuals diagnosed with EAC.

Strategies to identify and edit improvements in synthetic genome segments episomally.

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402-47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33-61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on therecoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

Exploring 3D community inconsistency in human chromosome contact networks

Abstract Researchers have developed chromosome capture methods such as Hi-C to better understand DNA’s 3D folding in nuclei. The Hi-C method captures contact frequencies between DNA segment pairs across the genome. When analyzing Hi-C data sets, it is common to group these pairs using standard bioinformatics methods (e.g. PCA). Other approaches handle Hi-C data as weighted networks, where connected node pairs represent DNA segments in 3D proximity. In this representation, one can leverage community detection techniques developed in complex network theory to group nodes into mesoscale communities containing nodes with similar connection patterns. While there are several successful attempts to analyze Hi-C data in this way, it is common to report and study the most typical community structure. But in reality, there are often several valid candidates. Therefore, depending on algorithm design, different community detection methods focusing on slightly different connectivity features may have differing views on the ideal node groupings. In fact, even the same community detection method may yield different results if using a stochastic algorithm. This ambiguity is fundamental to community detection and shared by most complex networks whenever interactions span all scales in the network. This is known as community inconsistency. This paper explores this inconsistency of 3D communities in Hi-C data for all human chromosomes. We base our analysis on two inconsistency metrics, one local and one global, and quantify the network scales where the community separation is most variable. For example, we find that TADs are less reliable than A/B compartments and that nodes with highly variable node-community memberships are associated with open chromatin. Overall, our study provides a helpful framework for data-driven researchers and increases awareness of some inherent challenges when clustering Hi-C data into 3D communities.

HLA-OLI: A new MHC class I pseudogene and HLA-Y are located on a 60 kb indel in the human MHC between HLA-W and HLA-J.

Analysis of publicly available whole-genome sequence data from the Human Pangenome Project and the 1000 Genomes Project has identified a DNA segment of approximately 60 kb in the major histocompatibility complex (MHC) between HLA-W and HLA-J that is present in some MHC haplotypes but not others. This DNA segment is largely repeat element-rich but includes the pseudogene HLA-Y, thus pinpointing the location of this pseudogene, and a new HLA class I sequence we have called HLA-OLI. HLA-OLI clusters phylogenetically with the HLA class I pseudogenes, HLA-P and HLA-W, and appears to have a similar genetic structure. The availability of whole-genome sequence data from diverse populations enables a detailed characterization of the MHC at the population level and will have implications for understanding MHC disease associations and the non-HLA MHC factors that impact unrelated hematopoietic cell transplant outcomes.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former’s inefficiency and the latter’s inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional ‘Correction’ mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in ‘Correction’ mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 (ND2) gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3′-CCAAACACAACTCCGAAAA-5′) and species-specific reverse primers composed of twenty (3′-CCAAACACAACTCCGAAAA-5′) and twenty-one (3′-TGGCAAGAATTAGGACGGTTA-5′) bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an in vitro experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that ND2 could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.