Unsupervised deep learning for identifying the O 6-carboxymethyl guanine by nanopore sequencing / 生物医学工程学杂志

O 6-carboxymethyl guanine(O 6-CMG) is a highly mutagenic alkylation product of DNA that causes gastrointestinal cancer in organisms. Existing studies used mutant Mycobacterium smegmatis porin A (MspA) nanopore assisted by Phi29 DNA polymerase to localize it. Recently, machine learning technology has been widely used in the analysis of nanopore sequencing data. But the machine learning always need a large number of data labels that have brought extra work burden to researchers, which greatly affects its practicability. Accordingly, this paper proposes a nano-Unsupervised-Deep-Learning method (nano-UDL) based on an unsupervised clustering algorithm to identify methylation events in nanopore data automatically. Specially, nano-UDL first uses the deep AutoEncoder to extract features from the nanopore dataset and then applies the MeanShift clustering algorithm to classify data. Besides, nano-UDL can extract the optimal features for clustering by joint optimizing the clustering loss and reconstruction loss. Experimental results demonstrate that nano-UDL has relatively accurate recognition accuracy on the O 6-CMG dataset and can accurately identify all sequence segments containing O 6-CMG. In order to further verify the robustness of nano-UDL, hyperparameter sensitivity verification and ablation experiments were carried out in this paper. Using machine learning to analyze nanopore data can effectively reduce the additional cost of manual data analysis, which is significant for many biological studies, including genome sequencing.

Application of nanopore sequencing in environmental microbiology research / 生物工程学报

The development of high-throughput sequencing techniques enabled a deeper and more comprehensive understanding of environmental microbiology. Specifically, the third-generation sequencing techniques represented by nanopore sequencing have greatly promoted the development of environmental microbiology research due to its advantages such as long sequencing reads, fast sequencing speed, real-time monitoring of sequencing data, and convenient machine carrying, as well as no GC bias and no PCR amplification requirement. This review briefly summarized the technical principle and characteristics of nanopore sequencing, followed by discussing the application of nanopore sequencing techniques in the amplicon sequencing, metagenome sequencing and whole genome sequencing of environmental microorganisms. The advantages and challenges of nanopore sequencing in the application of environmental microbiology research were also analyzed.

Research progress and application of nanopore sequencing technology / 生物工程学报

Sequencing technology has been greatly improved in terms of throughput and cost. The single-molecule nanopore DNA sequencing, one of the major branches of the third-generation sequencing technology, has made great contributions in the fields of medicine and life sciences due to its advantages of ultra-long reading length, real-time detection and direct detection of base methylation modification, etc. This article briefly describes the principle of nanopore sequencing technology, and discusses its application in clinical, animal, plant, bacterial and virus fields and its future development direction.

Analysis of unmapped regions associated with long deletions in Korean whole genome sequences based on short read data

While studies aimed at detecting and analyzing indels or single nucleotide polymorphisms within human genomic sequences have been actively conducted, studies on detecting long insertions/deletions are not easy to orchestrate. For the last 10 years, the availability of long read data of human genomes from PacBio or Nanopore platforms has increased, which makes it easier to detect long insertions/deletions. However, because long read data have a critical disadvantage due to their relatively high cost, many next generation sequencing data are produced mainly by short read sequencing machines. Here, we constructed programs to detect so-called unmapped regions (UMRs, where no reads are mapped on the reference genome), scanned 40 Korean genomes to select UMR long deletion candidates, and compared the candidates with the long deletion break points within the genomes available from the 1000 Genomes Project (1KGP). An average of about 36,000 UMRs were found in the 40 Korean genomes tested, 284 UMRs were common across the 40 genomes, and a total of 37,943 UMRs were found. Compared with the 74,045 break points provided by the 1KGP, 30,698 UMRs overlapped. As the number of compared samples increased from 1 to 40, the number of UMRs that overlapped with the break points also increased. This eventually reached a peak of 80.9% of the total UMRs found in this study. As the total number of overlapped UMRs could probably grow to encompass 74,045 break points with the inclusion of more Korean genomes, this approach could be practically useful for studies on long deletions utilizing short read data.

Rapid Whole-genome Sequencing of Zika Viruses using Direct RNA Sequencing

Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.

MinION(TM): New, Long Read, Portable Nucleic Acid Sequencing Device

The MinION(TM) is a miniature nanopore-based analysis device in which the characteristics of an analyte, as it passes through the nanopore, cause changes in the flow of ions through the pore, which are measured, as current flow, by a low noise amplifier and analogue-to-digital converter. Potentially any molecular analyte capable of passing through the nanopore may modify the flow of ions and generate a signal which might be diagnostic. In practice the current device is focussed on DNA sequencing, directly sequencing RNA is a likely development. With the MinION Access Program making the MinION(TM) widely available a flood of applications exploiting its real time, long read capabilities have been published. We review the background to the technology and compare it to current next generation sequencing.

[ effects of cell phone electromagnetic fields on the behavior of single ompf nanochannel forming protein: a practical approach]

Widespread of telecommunication systems in recent years, have raised the concerns on the possible danger of cell phone radiations on human body. Thus, the study of the electromagnetic fields on proteins, particularly the membrane nano channel forming proteins is of great importance. These proteins are responsible for keeping certain physic-chemical condition within cells and managing cell communication. Here, the effects of cell phones radiation on the activity of a single nanopore ion channel forming protein, OmpF, have been studied biophysically. Planar lipid bilayers were made based on Montal and Muller technique, and the activity of single OmpF channel reconstituted by electrical shock was recorded and analyzed by means of voltage-clamp technique at 20[degree]C. The planar lipid bilayers were formed from the monolayers made on a 60 micro m diameter aperture in the 20 micro m thick Teflon film that separated two [cis and trans] compartments of the glass chamber. In this practical approach we were able to analyze characteristics of an individual channel at different chemical and physical experimental conditions. The voltage clamp was used to measure the channel's conductance, voltage sensitivity, gating patterns in time scales as low as microseconds in real time. Our results showed that exposure of single voltage dependent channel, OmpF, to EMF of cell phone at high-frequency has a significant influence on the voltage sensitivity, gating properties and substate numbers of the single channel but has no effect on single-channel conductance. Regarding to the relaxation time, the channel also recovers in the millisecond time range when the field is removed. We observed an increase in the voltage sensitivity of the OmpF single channel while it had no effect on the single-channel conductance, which is remained to be further elucidated.

Molecular Methods for Studying the Human Microbiota

Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are important in investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity by selecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysis of complex human microbiota by culture-independent methods. Here, we will discuss features of tools for human microbiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategies that are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platforms such as PacBio and Oxford Nanopore are also introduced.

Irreversible Electroporation: A Novel Image-Guided Cancer Therapy

Irreversible electroporation (IRE) is a novel tumor ablation technique using a non-thermal energy to create innumerable permanent nanopores in the cell membrane to disrupt cellular homeostasis. This disruption of cellular homeostasis initiates apoptosis which leads to permanent cell death. In our translational research, we have demonstrated that IRE can be a safe, fast and powerful method of tumor treatment. In this review, we present an overview of IRE ablation including a brief history of IRE, advantages and disadvantages of IRE and clinical and research implications of IRE.