Short Fragments Research Articles

Accuracy of a simplified sequencing and bioinformatics platform on genetic variant detection in patients with prostate cancer.

e17061 Background: Consensus guidelines suggest that genetic testing is clinically indicated for prostate cancer (PCa) patients who meet certain family history, stage and histopathological grade criteria. Current next generation sequencing (NGS) diagnostics rely on capital and maintenance intensive sequencers which require highly trained technical personnel to operate. This may restrict availability of testing where specimen transport or access to personnel/capital is limited. We sought to compare results obtained using a simpler sequencer coupled with a bioinformatics platform which requires minimal maintenance and capital cost with those from more traditional NGS equipment. Methods: We compared results in detection of 10 genes recommended by NCCN PCa guidelines (ATM, BRCA1, BRCA2, CHEK2, MLH1, MSH2, MSH6, PALB2, PSM2, and HOXB13) between the Oxford Nanopore Technologies MinION to the Illumina NextSeq sequencer. All samples were sequenced in a depth greater than 50x. Bioinformatics for the Illumina sequencer was performed using Illumina’s TruSight Hereditary Cancer panel. As the MinION reads long DNA fragments, and cancer diagnostics is focused on short exon sequencing, we used the CorrSeq Dx by Foresee Genomics to create long DNA molecules consisting of several tandem repeats of short DNA fragments separated by sequences recognized by automated bioinformatics software. Statistical analysis was performed using Cohen’s kappa test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. Results: We detected using 380 variants in 13 patients on the Illumina platform and 379 (3 false negatives, 2 false positives) variants on the same patients using the MinION sequencer with CorrSeq Dx assay. The positive predictive value and sensitivity for the simplified platform was 99.5% (375/(375+2)) and 99.2% (375/(375+3)), respectively. The demonstrated accuracy of the simplified process was 99.9% (Cohen's kappa > 0.99). Conclusions: Thisl data suggests that a simpler sequencing solution with appropriate bioinformatics could allow clinical laboratories with limited technical and capital resources to perform germline and possibly somatic testing in-house; use of this technology may also serve to reduce costs for labs currently performing these tests. While some labs or clinics can outsource their NGS diagnostics if such commercial options are available, these options may not be possible where transport of specimens is logistically difficult or if access to technology is limited. A simpler process for NGS testing could improve access to guideline-based care in both remote and economically disadvantaged areas, resulting in better treatment outcomes as well as increased global healthcare equity. Further testing of this technology in prostate cancer as well as other disease states is warranted to fully understand its potential impact on patient care.

Using cell-free DNA to predict response to immunotherapy in non-small cell lung cancer pre- and post-treatment.

e21198 Background: Despite revolutionizing cancer therapy, immune checkpoint inhibitors (ICI) still do not benefit a significant proportion of patients. The risks associated with ICI-related adverse events, mixed performance of PD-L1 staining in predicting treatment response and its high cost, present a clinical need for more precise methods to define disease states in the context of ICI treatment. ICI response is thought to depend on the phenotype of the tumor and the associated immune response, especially functional state of CD8+ T cells in the tumor microenvironment (TME). Methods: Here we show that the CD8+ T cell signatures from plasma cell-free DNA (cfDNA) could be used to predict response of patients to anti-PD-1 therapy, using samples collected before and shortly after start of treatment. Blood samples were drawn just before the first dose, 1 day to 1 week before the start of treatment and 45-60 days since start of treatment. The treatment duration varied depending on response. Response was evaluated by CT scans every 8-12 weeks. 11 of the samples are from patients with no or minor response ( < 6 months of treatment), and 12 are from patients with prolonged benefit of the medication ( > 1 year of treatment). Exhausted CD8+ T cells, one of the targets of immunotherapy treatment are epigenetically regulated. So, we utilized naïve and PD-1high CD8+ T cell ATAC-seq data to define the repertoire of accessible chromatin specific to these cell types. We found an enrichment of short cfDNA fragments at a significant fraction of these sites, enabling us to define responder-specific and non-responder-specific accessible PD-1high regions from cfDNA. Results: We used the enrichment of short- versus long-cfDNA fragments (reflecting transcription factor-nucleosome dynamics in tissue of origin of cfDNA) as a scoring function, and repeated-cross-validation as a classifier model to identify differentially enriched features between responders and non-responders. Our model accurately predicts the two classes of response both pre-treatment (mean test AUC = 0.86; SD = 0.14). Conclusions: Notably, in addition to generating an accurate classifier, our analysis enabled us to identify predominant transcription factor motifs from predictive ATAC-seq peaks that characterize both response and lack of response, paving the way for further understanding the mechanistic basis of patient-specific response to ICI. Our results suggest the possibility of personalized prediction of treatment response that is independent of specific tumor genotype.

Global circulating free DNA methylation and fragmentome deconvolution in patients with metastatic renal cell carcinoma treated with immunotherapy (GOLDEN).

4544 Background: Metastatic renal cell carcinoma (mRCC) lacks molecular biomarkers. Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) is a new, non-invasive approach to the detection of RCC-derived DNA in the circulation. In the GOLDEN study (NCT03702309), we explored the role of cfMeDIP-seq and fragmentomic dynamics as a biomarker, during immune checkpoint inhibition (ICI)-based treatment in mRCC patients (pts). Methods: Blood samples from mRCC pts were collected before and during treatment. Response to treatment was defined by the treating oncologist as: response, any degree of radiological tumor regression; stable disease (SD), no radiological change; or progressive disease (PD), radiological/clinical progression. More than 10ng of plasma cfDNA was subject to cfMeDIP-seq. A prognostic pan-cancer methylation signature previously generated using TCGA tissue methylation arrays was applied to cfMeDIP-seq output and cancer-signal methylation (CSM) score was calculated. An RCC-specific methylation signature, previously generated by identifying differentially methylated regions between RCC pts and controls, was applied ( Nuzzo et al, Nat Med 2020). Genome-wide fragmentation profiles were generated using DELFI. Using medians as cut off, log-rank test was performed to assess overall survival (OS) and progression-free survival (PFS). Results: Thirty-five patients were enrolled and n=33 received systemic treatment. 31/35 pts (89%) had clear cell and 4/35 (11%) non-clear cell histology. Median age was 61 (30-81) years and the M:F ratio was 6:1. Patients’ IMDC prognostic risk score was favorable n=6/35 (17%), intermediate n=23/35 (65%) and poor n=6/35 (17%). 20/33 (61%) patients received ipilimumab/nivolumab, 10/33 (30%) nivolumab and n=3/33 (9%) pembrolizumab/axitinib. There were n=14/33 (42%) responders, n=4/33 (12%) pts with SD and 15/33 (45%) pts with PD. Patients with high pan-cancer CSM score at baseline trended towards worse OS (HR 3.86, p = 0.079). Fragmentomics signature, consisting of increased proportion of short cfDNA fragments, showed a trend for worse PFS (HR = 3.04, p = 0.075) in on-treatment samples. Using the RCC-specific methylation signature, we found that those with an increase in signature score from baseline to on-treatment had better PFS (HR=2.4x10-10, p=0.002, n=10) and a trend towards better OS (HR=0.17, p=0.074, n=10). We hypothesize that RCC tissue-specific cell death early during ICI treatment could underpin this increase in the methylation score. Conclusions: This small study is the first report on the role of cfMeDIPseq-derived methylation and fragmentomic signatures in patients with mRCC undergoing ICI-based treatment. We found several trends and one significant association between these signatures and pt outcomes, which support further validation in larger cohorts. Clinical trial information: NCT03702309 .

Pan-cancer detection and tissue-of-origin identification by whole-genome sequencing of plasma cell-free DNA.

3055 Background: Despite notable advances in cancer therapeutics, much of the mortality of human cancers results from late diagnosis. The early detection of cancer remains an unmet need that can benefit the effective treatments and better prognosis. Cell-free DNA (cfDNA) in the circulation provides an emerging diagnostic method for non-invasive cancer screening. The fragmentation signatures of cfDNA originating from different cell types or malignancies display distinct patterns, suggesting the potential as cancer biomarkers. This study aims to investigate the fragmentation profiles of cfDNA in several cancer types, and to develop a new approach for pan-cancer detection and tissue-of-origin identification. Methods: Plasma samples from 739 patients with lung (N=577), colorectal (N=98), breast (N=51) and liver (N=13) cancers, as well as 716 healthy individuals were collected in this study. Cell-free DNA was isolated and used to generate the whole-genome sequencing data. We developed an approach called PatternWGS (Pan-cancer detection and tissue-of-origin identification by whole-genome sequencing) to analyze the DNA fragmentation patterns comprehensively. Fragmentation features integrating cfDNA fragment size distribution, frequency of end motif sequence, and copy number variation were utilized to construct the optimal prediction model. We presented a multi-cancer detection model for distinguishing individuals with and without cancer using Generalized Linear Models (glmnet), and further built a cancer-origin model to predict cancer types using Random Forest algorithm. Training and testing occurred on randomly selected sampling splits of 60% and 40% of the data, respectively. Features selection and model construction were performed by only training cohort, and the test set was solely used for performance evaluation. Results: A larger proportion of shorter cfDNA fragments in cancers was observed. We proposed a fragment score indicating the degree of short fragmentation, and this fragment score was significantly higher in cancers (p<0.001). Several end motifs were identified to be specifically enriched in cancer patients. The multi-cancer detection model reached an area under the curve (AUC) of 0.98, with the sensitivity of 93% at 95% specificity. The sensitivity of detecting lung, colorectal, breast, and liver cancers reached 94.2%, 87%, 100% and 100% respectively, at 95% specificity. The cancer-origin model demonstrated an overall accuracy of 81.7% for the identification of tissue of origin. Conclusions: Our prediction model demonstrated a superior performance for multi-cancer detection and cancer origin prediction, suggesting the potential of plasma cfDNA fragmentation signatures as non-invasive biomarkers for early cancer detection in clinical. The distinct fragmentation patterns in cancer provide new insight to understand the mechanism of tumorigenesis.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

A rapid protocol for ribosome profiling of low input samples.

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

Genetic variability and mutation of Epstein‒Barr virus (EBV)-encoded LMP-1 and BHRF-1 genes in EBV-infected patients: identification of precise targets for development of personalized EBV vaccines.

The critical Epstein‒Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) and BamHI fragment H rightward open reading frame 1 (BHRF-1) genes affect EBV-mediated malignant transformation and virus replication during EBV infection. Therefore, these two genes are considered ideal targets for EBV vaccine development. However, gene mutations in LMP-1 and BHRF-1 in different cohorts may affect the biological functions of EBV, which would seriously hinder development of personalized vaccines for EBV. In the present study, by performing nested polymerase chain reaction (nested PCR) and DNA sequence techniques, we analyzed the nucleotide variability and phylogeny of LMP-1 containing a 30bp deletion region (del-LMP-1) and BHRF-1 in EBV-infected patients (N = 382) and healthy persons receiving physical examination (N = 98; defined as the control group) in Yunnan Province, China. Three BHRF-1 subtypes were identified in this study: 79V88V, 79L88L, and 79V88L, with mutation frequencies of 58.59%, 24.24%, and 17.17%, respectively. Compared with the control group, the distribution of BHRF-1 subtypes of the three groups showed no significant difference, suggesting that BHRF-1 is highly conserved in EBV-related samples. In addition, a short fragment of del-LMP-1 was found in 133 cases, and the nucleotide variation rate was 87.50% (133/152). For del-LMP-1, a significant distribution in three groups was detected, as characterized by a high mutation rate. In conclusion, our study illustrates gene variability and mutations of EBV-encoded del-LMP-1 and BHRF-1 in clinical samples. Highly mutated LMP-1 might be associated with various types of EBV-related diseases, indicating that BHRF-1 combined with LMP-1 may be used as an ideal target for development of EBV personalized vaccines.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

Associations between circulating cell-free mitochondrial DNA, inflammatory markers, and cognitive and physical outcomes in community dwelling older adults

BackgroundDementia and frailty are common age-related syndromes often linked to chronic inflammation. Identifying the biological factors and pathways that contribute to chronic inflammation is crucial for developing new therapeutic targets. Circulating cell-free mitochondrial DNA (ccf-mtDNA) has been proposed as an immune stimulator and potential predictor of mortality in acute illnesses. Dementia and frailty are both associated with mitochondrial dysfunction, impaired cellular energetics, and cell death. The size and abundance of ccf-mtDNA fragments may indicate the mechanism of cell death: long fragments typically result from necrosis, while short fragments arise from apoptosis. We hypothesize that increased levels of necrosis-associated long ccf-mtDNA fragments and inflammatory markers in serum are linked to declines in cognitive and physical function, as well as increased mortality risk.ResultsOur study of 672 community-dwelling older adults revealed that inflammatory markers (C-Reactive Protein, soluble tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 [sTNFR1], and interleukin-6 [IL-6]) positively correlated with ccf-mtDNA levels in serum. Although cross-sectional analysis revealed no significant associations between short and long ccf-mtDNA fragments, longitudinal analysis demonstrated a connection between higher long ccf-mtDNA fragments (necrosis-associated) and worsening composite gait scores over time. Additionally, increased mortality risk was observed only in individuals with elevated sTNFR1 levels.ConclusionIn a community dwelling cohort of older adults, there are cross-sectional and longitudinal associations between ccf-mtDNA and sTNFR1 with impaired physical and cognitive function and increased hazard of death. This work suggests a role for long ccf-mtDNA as a blood-based marker predictive of future physical decline.

A Newly Identified Spike Protein Targeted Linear B-Cell Epitope Based Dissolvable Microneedle Array Successfully Eliciting Neutralizing Activities against SARS-CoV-2 Wild-Type Strain in Mice.

Vaccination is a cost-effective medical intervention. Inactivated whole virusor large protein fragments-based severe acute respiratory syndrome coronavirus (SARS-CoV-2) vaccines have high unnecessary antigenic load to induce allergenicity and/orreactogenicity, which can be avoided by peptide vaccines of short peptide fragments that may induce highly targeted immune response. However, epitope identification and peptide delivery remain the major obstacles in developing peptide vaccines. Here, a multi-source data integrated linear B-cell epitope screening strategy is presented and a linear B-cell epitope enriched hotspot region is identified in Spike protein, from which a monomeric peptide vaccine (Epitope25) is developed and applied to subcutaneously immunize wildtype BALB/c mice. Indirect ELISA assay reveals specific and dose-dependent binding between Epitope25 and serum IgG antibodies from immunized mice. The neutralizing activity of sera from vaccinated mice is validated by pseudo and live SARS-CoV-2 wild-type strain neutralization assays. Then a dissolvable microneedle array (DMNA) is developed to pain-freely deliver Epitope25. Compared with intramuscular injection, DMNA and subcutaneous injection elicit neutralizing activities against SARS-CoV-2 wild-type strain as demonstrated by live SARS-CoV-2 virus neutralization assay. No obvious damages are found in major organs of immunized mice. This study may lay the foundation for developing linear B-cell epitope-based vaccines against SARS-CoV-2.

Over 30-Fold Enhancement in DNA Translocation Dynamics through Nanoscale Pores Coated with an Anionic Surfactant.

Solid-state nanopores (ssNPs) are single-molecule sensors capable of label-free quantification of different biomolecules, which have become highly versatile with the introduction of different surface treatments. By modulating the surface charges of the ssNP, the electro-osmotic flow (EOF) can be controlled in turn affecting the in-pore hydrodynamic forces. Herein, we demonstrate that negative charge surfactant coating to ssNPs generates EOF that slows-down DNA translocation speed by >30-fold, without deterioration of the NP noise, hence significantly improving its performances. Consequently, surfactant-coated ssNPs can be used to reliably sense short DNA fragments at high voltage bias. To shed light on the EOF phenomena inside planar ssNPs, we introduce visualization of the electrically neutral fluorescent molecule's flow, hence decoupling the electrophoretic from EOF forces. Finite elements simulations are then used to show that EOF is likely responsible for in-pore drag and size-selective capture rate. This study broadens ssNPs use for multianalyte sensing in a single device.

Semiclassical treatment of Feshbach resonances by transfer matrices.

A semiclassical method is presented for the calculation of Feshbach resonance positions and widths. This approach, based on semiclassical transfer matrices, relies only on relatively short trajectory fragments, thus avoiding problems associated with the long trajectories needed in more straightforward semiclassical techniques. Complex resonance energies are obtained from an implicit equation that is developed to compensate for the inaccuracy of the stationary phase approximation underlying the semiclassical transfer matrix applications. Although this treatment requires calculation of transfer matrices for complex energies, an initial value representation method makes it possible to extract such quantities from ordinary real-valued classical trajectories. This treatment is applied to obtain positions and widths for resonances in a model two-dimensional system, and the results are compared to those obtained from accurate quantum mechanical calculations. The semiclassical method successfully captures the irregular energy dependence of resonance widths that vary over a range of more than two orders of magnitude. An explicit semiclassical expression for the width of narrow resonances is also presented and serves as a simpler, useful approximation for many cases.

Evaluation of microhaplotype panels for complex kinship analysis using massively parallel sequencing

In recent years, microhaplotypes (MHs) have become a research hotspot within the field of forensic genetics. Traditional MHs contain only SNPs that are closely linked within short fragments. Herein, we broaden the concept of general MHs to include short InDels. Complex kinship identification plays an important role in disaster victim identification and criminal investigations. For distant relatives (e.g., 3rd-degree), many genetic markers are required to enhance power of kinship testing. We performed genome-wide screening for new MH markers composed of two or more variants (InDel or SNP) within 220 bp based on the Chinese Southern Han from the 1000 Genomes Project. An NGS-based 67plex MH panel (Panel B) was successfully developed, and 124 unrelated individual samples were sequenced to obtain population genetic data, including alleles and allele frequencies. Of the 67 genetic markers, 65 MHs were, as far as we know, newly discovered, and 32 MHs had effective number of allele (Ae) values greater than 5.0. The average Ae and heterozygosity of the panel were 5.34 and 0.7352, respectively. Next, 53 MHs from a previous study were collected as Panel A (average Ae of 7.43), and Panel C with 87 MHs (average Ae of 7.02) was formed by combining Panels A and B. We investigated the utility of these three panels in kinship analysis (parent-child, full siblings, 2nd-degree, 3rd-degree, 4th-degree, and 5th-degree relatives), with Panel C exhibiting better performance than the two other panels. Panel C was able to separate parent-child, full-sibling, and 2nd-degree relative duos from unrelated controls in real pedigree data, with a small false testing level (FTL) of 0.11% in simulated 2nd-degree duos. For more distant relationships, the FTL was much higher: 8.99% for 3rd-degree, 35.46% for 4th-degree, and 61.55% for 5th-degree. When a carefully chosen extra relative was known, this may enhance the testing power for distant kinship analysis. Two twins from the Q family (2–5 and 2–7) and W family (3–18 and 3–19) shared the same genotypes in all tested MHs, which led to the incorrect conclusion that an uncle-nephew duo was classified as a parent-child duo. In addition, Panel C showed great capacity for excluding close relatives (2nd-degree and 3rd-degree relatives) during paternity tests. Among 18,246 real and 10,000 simulated unrelated pairs, none were misinterpreted as a relative within 2nd-degree at a log10(LR) cutoff of 4. The panels presented herein could provide supplementary power for the analysis of complex kinship.

Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28°C for times ranging from 0 to 120h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60h), whereas the medium and longest loci were not amplified by conventional PCR even at 0h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

Variants detection in regions of segmental duplication facilitates molecular diagnosis for IEI such as chronic granulomatous disease caused by NCF1 mutations

With the expanding application of next-generation sequencing (NGS) in clinical practice, rapid progress has been made in molecular diagnoses for diseases such as inborn errors of immunity (IEI). However, due to characteristic short read length and fragment size for NGS, genomic regions with segmental duplication pose a big challenge for accurate mutation detection. Reads originating from segmental duplications are mapped with high ambiguity and are often discarded by most variant callers, leading to poor variant identification. Dozens of known IEI causal genes are located in regions of segmental duplications, such as NCF1, IGLL1, FCGR3A, etc. It is important to increase the sensitivity of mutation detections in such regions.In this study, we develop a strategy to significantly increase the sensitivity of identifying rare germline variants in regions of segmental duplication for IEI patients while controlling the number of false positives. By forcing multi-aligned reads from segmental duplications to map to functionally significant coding regions in the region concerned, all possible small variants are being identified. This is followed by the identification of false positives originating from intrinsic sequence differences among genomic duplications and the identification of potential common variants by matching with an in-house database.Using simulated data and real-world data from the 'Genome In a Bottle’ project, we demonstrate a significant improvement in variant detection sensitivity, ranging from 80% to 98% and 75% to 94%, respectively. This is accompanied by a limited increase in false positives that can be dealt with by downstream analysis. Applying our method to whole exome sequencing data led to the identification of the common ΔGT mutation in NCF1 in three patients with the chronic granulomatous disease, demonstrating the power of this novel approach.In summary, we developed SDrecall, a bioinformatic method that is by far the most robust open-source approach to detect rare, disease-causing variants in regions of segmental duplication. The method should apply to both IEI and other Mendelian diseases with causal mutations located in such regions and is suited to WGS, WES, and targeted sequencing data.

Balancing read length and sequencing depth: Optimizing Nanopore long-read sequencing for monocots with an emphasis on the Liliales.

We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results. Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.

Co‐Assembly of Carbon Nanotube Porins into Biomimetic Peptoid Membranes (Small 21/2023)

Biomimetic Peptoid Membranes Co-assembly of carbon nanotube porins into peptoid sheets creates novel precision pore membrane nanostructures. The membrane matrix is made up of peptoids, self-assembled designed sequence polymers, while carbon nanotube porins, short fragments of carbon nanotubes inserted into the peptoid matrix, form precision pores. This new membrane could be advantageous for high-value precision separation applications. More details can be found in article number 2206810 by Marcel Baer, Aleksandr Noy, Chun-Long Chen, and co-workers.

Prediction of miRNA-disease associations in microbes based on graph convolutional networks and autoencoders.

MicroRNAs (miRNAs) are short RNA molecular fragments that regulate gene expression by targeting and inhibiting the expression of specific RNAs. Due to the fact that microRNAs affect many diseases in microbial ecology, it is necessary to predict microRNAs' association with diseases at the microbial level. To this end, we propose a novel model, termed as GCNA-MDA, where dual-autoencoder and graph convolutional network (GCN) are integrated to predict miRNA-disease association. The proposed method leverages autoencoders to extract robust representations of miRNAs and diseases and meantime exploits GCN to capture the topological information of miRNA-disease networks. To alleviate the impact of insufficient information for the original data, the association similarity and feature similarity data are combined to calculate a more complete initial basic vector of nodes. The experimental results on the benchmark datasets demonstrate that compared with the existing representative methods, the proposed method has achieved the superior performance and its precision reaches up to 0.8982. These results demonstrate that the proposed method can serve as a tool for exploring miRNA-disease associations in microbial environments.

Development of a high-resolution mass-spectrometry-based method and software for human leukocyte antigen typing.

The human leukocyte antigen (HLA) system plays a critical role in the human immune system and is strongly associated with immune recognition and rejection in organ transplantation. HLA typing method has been extensively studied to increase the success rates of clinical organ transplantation. However, while polymerase chain reaction sequence-based typing (PCR-SBT) remains the gold standard, cis/trans ambiguity and nucleotide sequencing signal overlay during heterozygous typing present a problem. The high cost and low processing speed of Next Generation Sequencing (NGS) also render this approach inadequate for HLA typing. To address these limitations of the current HLA typing methods, we developed a novel typing technology based on nucleic acid mass spectrometry (MS) of HLA. Our method takes advantage of the high-resolution mass analysis function of MS and HLAMSTTs (HLA MS Typing Tags, some short fragment PCR amplification target products) with precise primer combinations. We correctly typed HLA by measuring the molecular weights of HLAMSTTs with single nucleotide polymorphisms (SNPs). In addition, we developed a supporting HLA MS typing software to design PCR primers, construct the MS database, and select the best-matching HLA typing results. With this new method, we typed 16 HLA-DQA1 samples, including 6 homozygotes and 10 heterozygotes. The MS typing results were validated by PCR-SBT. The MS HLA typing method is rapid, efficient, accurate, and readily applicable to typing of homozygous and heterozygous samples.