Lukas Zemaitis

DNA data storage allows sequences to be defined without biological constraints, yet readout workflows still depend on generic end-repair/dA-tailing chemistry. We developed NinjaSeq, a type IIS restriction endonuclease library-preparation strategy that incorporates recognition sites into primer flanks, enabling digestion to generate adapter-compatible overhangs and eliminating the need for conventional end preparation. By combining this chemistry with constrained coding that excludes internal recognition motifs, NinjaSeq produced sequencing quality and decoding performance consistent with standard protocols while reducing reagent burden and simplifying processing, including compatibility with one-pot restriction-ligation. The same sequence-directed design also enables physical random access during library preparation: targeting file-specific flanking sites enriched a desired file from a mixed pool by about sixteen-fold in a proof-of-concept experiment. These results position NinjaSeq as a practical ONT readout approach for DNA data storage.

In recent years, Oxford Nanopore Technologies (ONT) has gained substantial attention across various domains of nucleic acid research, owing to its unique advantages over other sequencing platforms. Originally developed for long-read sequencing, ONT technology has evolved, with recent advancements enhancing its applicability beyond long reads to include short, synthetic DNA-based applications. However, sequencing short DNA fragments with nanopore technology often results in lower data quality, likely due to the absence of protocols optimised for these fragment sizes. To address this challenge, we refined the standard ONT library preparation protocol to improve its performance for ultra-short DNA targets. By utilising the same core reagents required for conventional ONT workflows, we introduced targeted alterations to enhance compatibility with shorter fragment lengths. We then benchmarked these adjustments against libraries prepared using the standard ONT protocol. Here, we present a comprehensive, step-by-step protocol that is accessible to researchers of various technical expertise, facilitating high-quality sequencing of ultra-short DNA fragments. This protocol represents a significant improvement in sequencing quality for short DNA sequences using ONT technology, broadening the range of possible applications.

Background and Objective: Unexplained infertility is a major challenge in reproductive medicine and requires advanced diagnostic approaches to identify the underlying factors accurately. This study aims to evaluate the utility of risk factor analysis and a gene panel in diagnosing unexplained infertility using the next-generation sequencing (NGS) technology. Our study aimed to characterize and identify risk and genetic factors associated with unexplained infertility. Materials and methods: A cohort of patients with unexplained infertility was comprehensively screened for risk factors and genetic variations using a targeted gene panel (10 couples with unexplained infertility (UI) and 36 fertile couples). 108 articles were selected (58 on female infertility and 50 on male infertility) presenting genes that may be associated with unexplained infertility. A gene panel for unexplained infertility was compiled based on the literature data. A customized virtual panel was created from the exome sequencing data. Results: In the female group, controls had a higher mean age, while in the male patients, both groups were similar in terms of age. Both gender groups had comparable BMI values. No significant associations (p > 0.05) between risk factors and unexplained infertility were found when evaluating anthropometric parameters and other sociodemographic characteristics. In two male patients (20%), a molecular defect was detected in NGS variants classified aspossible benign and probably benign In particular, missense variants were identified in the UGT2B7 and CATSPER2 genes, A molecular defect classified as probably damaging was found in five female patients (50%). In particular, missense variants were identified in the CAPN10, MLH3, HABP2, IRS1, GDF9, and SLC19A1 genes. Conclusions: The study emphasizes that unexplained infertility is often related to mechanisms beyond causative mutations and highlights the need for integrative genomic research involving broader gene panels and multi-faceted approaches, including transcriptomics and epigenetics, to uncover latent genetic predispositions.

In recent years, Oxford Nanopore Technologies (ONT) has gained substantial attention across various domains of nucleic acids’ research, owing to its unique advantages over other sequencing platforms. Originally developed for long-read sequencing, ONT technology has evolved, with recent advancements enhancing its applicability beyond long reads to include short, synthetic DNA-based applications. However, sequencing short DNA fragments with nanopore technology often results in lower data quality, likely due to a lack of protocols optimised for these fragment sizes. To address this challenge, we refined the standard ONT library preparation protocol to improve its performance for ultra-short DNA targets. Utilising the same core reagents required for conventional ONT workflows, we introduced targeted alterations to enhance compatibility with shorter fragment lengths. We then benchmarked these adjustments against libraries prepared using the standard ONT protocol. Here, we present a comprehensive, step-by-step protocol that is accessible to researchers of varied technical expertise, facilitating high-quality sequencing of ultra-short DNA fragments. This protocol represents a significant improvement in sequencing quality for short DNA fragments using ONT technology, broadening the range of possible applications. Graphical

Here, we report the emergence of the variant lineage B.1.1.523 that contains a set of mutations including 156_158del, E484K and S494P in the spike protein. E484K and S494P are known to significantly reduce SARS-CoV-2 neutralization by convalescent and vaccinated sera and are considered as mutations of concern. Lineage B.1.1.523 presumably originated in the Russian Federation and spread across European countries with the peak of transmission in April–May 2021. The B.1.1.523 lineage has now been reported from 31 countries. In this article, we analyze the possible origin of this mutation subset and its immune response using in silico methods.

SARS-CoV-2 has spread vastly throughout the word. In this study, we focus on the patterns of spread in Lithuania. By analysing the genetically sequenced data of different lineages and their first appearances, we were able to compare the dynamics of spreading of the lineages and recognize the main possible cause. The impact of emigration patterns and international travel on the variety of lineages was also assessed. Results showed different patterns of spread, and while a vast variety of different lineages were brought in by international travel, many of the viral outbreaks were caused by local lineages. It can be concluded that international travel had the most impact on the spread of SARS-CoV-2.

Background. Evidence shows that microRNAs (miRNAs) could play a key role in the homeostasis and development of major depressive disorder and bipolar disorder. The present study is aimed at investigating the changes in circulating miRNA expression profiles in a plasma of patients suffering from major depressive disorder (MDD) and bipolar disorder (BD) to distinguish and evaluate these molecules as biomarkers for mood disorders. Methods. A study enrolled a total of 184 subjects: 74 controls, 84 MDD patients, and 26 BD patients. Small RNA sequencing revealed 11 deregulated circulating miRNAs in MDD and BD plasma, of which expression of 5, hsa-miR-139-3p, miRNAs hsa-let-7e-5p, hsa-let-7f-5p, hsa-miR-125a-5p, and hsa-miR-483-5p, were further verified using qPCR. miRNA gene expression data was evaluated alongside the data from clinical assessment questionnaires. Results. hsa-let-7e-5p and hsa-miR-125a-5p were both confirmed upregulated: 0.75-fold and 0.25-fold, respectively, in the MDD group as well as 1.36-fold and 0.68-fold in the BD group. Receiver operating curve (ROC) analysis showed mediocre diagnostic sensitivity and specificity of both hsa-let-7e-5p and hsa-miR-125a-5p with approximate area under the curve (AOC) of 0.66. ROC analysis of combined miRNA and clinical assessment data showed that hsa-let-7e-5p and hsa-miR-125a-5p testing could improve MDD and BD diagnostic accuracy by approximately 10. Conclusions. Circulating hsa-let-7e-5 and hsa-miR-125a-5p could serve as additional peripheral biomarkers for mood disorders; however, suicidal ideation remains the major diagnostic factor for MDD and BD.

Here we report the emergence of variant lineage B.1.1.523 that contains a set of mutations including 156_158del, E484K and S494P in Spike protein. E484K and S494P are known to significantly reduce SARS-CoV-2 neutralization by convalescent and vaccinee sera and are considered as mutations of concern. Lineage B.1.1.523 has presumably originated in Russian Federation and spread across European countries with the peak of transmission in April – May 2021. The B.1.1.523 lineage has now been reported from 27 countries.