Single-molecule sequencing (SMS), a long-read DNA sequencing technology, plays a crucial role in genomics research. However, traditional ultrasonic shearing techniques struggle to efficiently produce DNA fragments ≥10 kbp, limiting the efficiency of SMS library preparation. Here, we developed a zirconia bead-mediated ultrasonic shearing method that enables precise DNA fragmentation through zirconia bead mechanical agitation induced by sonication cavitation. By optimizing parameters such as zirconia bead size, quantity, ultrasonic probe distance, ultrasonic time, water bath temperature, DNA sample volume, and DNA concentration, we obtained target fragments in the 10-20 kbp range. The results demonstrated that this method sheared purified λDNA (48.5 kbp) into fragments averaging 15 kbp within 20 seconds, achieving performance comparable to commercial g-TUBE methods. The method was also successfully applied to human genomic DNA. This simple, rapid and reliable DNA fragmentation method provides an effective solution for SMS library preparation with great potential for molecular detection and diagnostic applications.

We describe a method for fragmenting, in-situ, surface-adsorbed and immobilized DNAs on polymethylmethacrylate(PMMA)-coated silicon substrates using microfluidic delivery of the cutting enzyme DNase I. Soft lithography is used to produce silicone elastomer (Sylgard 184) gratings which form microfluidic channels for delivery of the enzyme. Bovine serum albumin (BSA) is used to reduce DNase I adsorption to the walls of the microchannels and enable diffusion of the cutting enzyme to a distance of 10mm. Due to the DNAs being immobilized, the fragment order is maintained on the surface. Possible methods of preserving the order for application to sequencing are discussed.

Oral microbiome plays a key role in the etiology of oral diseases and is linked to many diseases in other parts of the body as well. This makes the oral microbiome an area of interest for researchers globally. A meticulous planning of the research project is the first and most crucial step while conducting an oral microbiome study. For beginners in this field, it is essential to be familiar with the terminologies used in oral microbiome research for a better understanding. The purpose of this article is to familiarize new researchers to the frequently used terms for the field of oral microbiome research.

Cutaneous melanoma is an aggressive tumor responsible for 90% of mortality related to skin cancer. In the recent years, the discovery of driving mutations in melanoma has led to better treatment approaches. The last decade has seen a genomic revolution in the field of cancer. Such genomic revolution has led to the production of an unprecedented mole of data. High-throughput genomic technologies have facilitated the genomic, transcriptomic and epigenomic profiling of several cancers, including melanoma. Nevertheless, there are a number of newer genomic technologies that have not yet been employed in large studies. In this article we describe the current classification of cutaneous melanoma, we review the current knowledge of the main genetic alterations of cutaneous melanoma and their related impact on targeted therapies, and we describe the most recent high-throughput genomic technologies, highlighting their advantages and disadvantages. We hope that the current review will also help scientists to identify the most suitable technology to address melanoma-related relevant questions. The translation of this knowledge and all actual advancements into the clinical practice will be helpful in better defining the different molecular subsets of melanoma patients and provide new tools to address relevant questions on disease management. Genomic technologies might indeed allow to better predict the biological - and, subsequently, clinical - behavior for each subset of melanoma patients as well as to even identify all molecular changes in tumor cell populations during disease evolution toward a real achievement of a personalized medicine.

Although technological advances have greatly reduced the cost of DNA sequencing, sample preparation time and reagent costs remain the limiting factors for many studies. Based on low-cost targeted amplification, we developed an economical method for custom target library construction based on DNA nanoball (DNB) technology and two-step polymerase chain reaction (PCR). Here, we refer to this method as the two-step PCR, which was compared to traditional multiplex PCR methods in three aspects, data quality, efficiency, and specificity to humans. The results confirmed that two-step PCR reduces to finishing 128 sequencing libraries in only 2 h 24 min 59 s of the total PCR time and at a data utilization rate of 0.44 at a cost of approximately $1.70 per sample for targeted sequencing via the two-step PCR. The replacement of traditional multiplex PCR methods with this strategy makes the sample preparation process before sequencing relatively more cost-effective and further reduces the cost of next-generation sequencing (NGS). This method may also be free from the interference of other species and the limitations of sample type and DNA content. These findings reveal possibilities for broad applications of this approach in forensic research.

Studies on foraging partitioning in pollinators can provide critical information to the understanding of food-web niche and pollination functions, thus aiding conservation. Metabarcoding based on PCR amplification and high-throughput sequencing has seen increasing applications in characterizing pollen loads carried by pollinators. However, amplification bias across taxa could lead to unpredictable artefacts in estimation of pollen compositions. We examined the efficacy of a genome-skimming method based on direct shotgun sequencing in quantifying mixed pollen, using mock samples (five and 14 mocks of flower and bee pollen, respectively). The results demonstrated a high level of repeatability and accuracy in identifying pollen from mixtures of varied species ratios. All pollen species were detected in all mocks, and pollen frequencies estimated from the number of sequence reads of each species were significantly correlated with pollen count proportions (linear model, R²  = 86.7%, p = 2.2e-16). For >97% of the mixed taxa, pollen proportion could be quantified by sequencing to the correct order of magnitude, even for species which constituted only 0.2% of the total pollen. In addition, DNA extracted from pollen grains equivalent to those collected from a single honeybee corbicula was sufficient for genome-skimming. We conclude that genome-skimming is a feasible approach to identifying and quantifying mixed pollen samples. By providing reliable and sensitive taxon identification and relative abundance, this method is expected to improve our understanding in studies that involve plant-pollinator interactions, such as pollen preference in corbiculate bees, pollen diet analyses and identification of landscape pollen resource use from beehives.

Current techniques for making high-resolution, photolithographic DNA microarrays suffer from the limitation that the 3' end of each sequence is anchored to a hard substrate and hence is unavailable for many potential enzymatic reactions. Here, we demonstrate a technique that inverts the entire microarray into a hydrogel. This method preserves the spatial fidelity of the original pattern while simultaneously removing incorrectly synthesized oligomers that are inherent to all other microarray fabrication strategies. First, a standard 5'-up microarray on a donor wafer is synthesized, in which each oligo is anchored with a cleavable linker at the 3' end and an Acrydite phosphoramidite at the 5' end. Following the synthesis of the array, an acrylamide monomer solution is applied to the donor wafer, and an acrylamide-silanized acceptor wafer is placed on top. As the polyacrylamide hydrogel forms between the two wafers, it covalently incorporates the acrydite-terminated sequences into the matrix. Finally, the oligos are released from the donor wafer upon immersing in an ammonia solution that cleaves the 3'-linkers, thus freeing the oligos at the 3' end. The array is now presented 3'-up on the surface of the gel-coated acceptor wafer. Various types of on-gel enzymatic reactions demonstrate a versatile and robust platform that can easily be constructed with far more molecular complexity than traditional photolithographic arrays by endowing the system with multiple enzymatic substrates. We produce a new generation of microarrays where highly ordered, purified oligos are inverted 3'-up, in a biocompatible soft hydrogel, and functional with respect to a wide variety of programable enzymatic reactions.