Junji Li

Systematic Characteristic Exploration of the Chimeras Generated in Multiple Displacement Amplification through Next Generation Sequencing Data Reanalysis

Background The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA) and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome. Method We finished data mining towards a series of Next Generation Sequencing (NGS) reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length), and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction. Results/Conclusion 131.4 Gb pair-end (PE) sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19%) with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a linear correlation analysis. Significance This study exhibited the profile of the phi29MDA chimeras by tens of millions of chimeric sequences, and helped understand the amplification mechanism of the phi29 DNA polymerase. Our work also illustrated the importance of NGS data reanalysis, not only for the improvement of data utilization efficiency, but also for more potential genomic information.

PDMS-based microfluidic devices using commoditized PCBs as masters with no specialized equipment required

Designed printed circuit boards (PCBs) are alternative substrates for master mold construction of microfluidic devices. However, the rough supportive material molds rough replicas and causes difficulty in device sealing. To overcome this difficulty, a copper layer is used to generate a smooth surface. Some other researchers have used thermoplastic elastomers, which are typically much easier to bond, instead of the general polymer polydimethysiloxane (PDMS). This study presents an extremely simple approach for fabricating PDMS-based microfluidic devices using PCBs as masters. Unlike those reported in the previous studies, commoditized PCBs fabricated at a common PCB manufactory were directly used as the master molds. Two layers of semi-cured silicone of a distinct base to curing agent ratios were bonded together by additional curing. Efficient bonding was accomplished and avoided insufficient adhesion due to the rough surfaces of the PDMS replicas. Highly monodisperse droplets with polydispersity values smaller than 1% were stably formed using the easy fabricated devices. Essential operations in droplet microfluidics were reliably conducted in the PDMS-based devices. Moreover, the droplets were orderly sorted by the microstructures in the fabricated multi-height devices. The fabrication process provided a simple, convenient and reliable approach to prepare the general polymer PDMS-based microfluidic devices with a minimal requirement for equipment.

A cell sorting and trapping microfluidic device with an interdigital channel

The growing interest in cell sorting and trapping is driving the demand for high performance technologies. Using labeling techniques or external forces, cells can be identified by a series of methods. However, all of these methods require complicated systems with expensive devices. Based on inherent differences in cellular morphology, cells can be sorted by specific structures in microfluidic devices. The weir filter is a basic and efficient cell sorting and trapping structure. However, in some existing weir devices, because of cell deformability and high flow velocity in gaps, trapped cells may become stuck or even pass through the gaps. Here, we designed and fabricated a microfluidic device with interdigital channels for cell sorting and trapping. The chip consisted of a sheet of silicone elastomer polydimethylsiloxane and a sheet of glass. A square-wave-like weir was designed in the middle of the channel, comprising the interdigital channels. The square-wave pattern extended the weir length by three ti...

1D-Reactor Decentralized MDA for Uniform and Accurate Whole Genome Amplification

Multiple displacement amplification (MDA), a most popular isothermal whole genome amplification (WGA) method, suffers the major hurdle of highly uneven amplification, thus, leading to many problems in approaching biological applications related to copy-number assessment. In addition to the optimization of reagents and conditions, complete physical separation of the entire reaction system into numerous tiny chambers or droplets using microfluidic devices, has been proven efficient to mitigate this amplifying bias in recent works. Here, we present another MDA advance, microchannel MDA (μcMDA), which decentralizes MDA reagents throughout a one-dimensional slender tube. Due to the double effect from soft partition of high molecular-weight DNA molecules and less-limited diffusion of small particles, μcMDA is shown to be significantly effective at improving the amplification uniformity, which enables us to accurately detect single nucleotide variants (SNVs) with higher efficiency and sensitivity. More importantly, this straightforward method requires neither customized instruments nor complicated operations, making it a ready-to-use technique in almost all biological laboratories.

Fabrication of a microfluidic chip based on the pure polypropylene material

Polypropylene (PP) material has been widely used in the biomedical field for decades due to its high reliability in biochemical reactions. However, a pure PP material microfluidic chip has rarely been reported. Herein, a simple and rapid method has been developed to fabricate a pure PP microfluidic chip by a thermal bonding process using a PP film and PP substrate. An experiment for two-temperature PCR in the pure PP microfluidic system without a pre-treatment process has been successfully carried out. It is shown that the PP microfluidic chip has a high structural strength, tightness for water sealing, and low nonspecific adsorption, which promotes the practical application of microfluidics in the biomedical field. Compared to other existing microfluidic chip technologies, our pure PP material microfluidic chip and its fabrication method have many advantages such as high-speed production rate and extremely low cost. It can be achieved in industrial assembly lines for standardized manufacturing.

The complete mitochondrial genome sequence of Boechera stricta

Abstract Boechera stricta (B. stricta) is a wild relative of Arabidopsis, occurring in mostly montane regions of western North America. In this article, we assembled the complete mitochondrial (mt) DNA sequence of B. stricta into a circular genome of length 271,601 bp, including 31 protein-coding genes, 21 tRNA genes, and 3 rRNA genes. From the neighbour-joining phylogenetic tree was constructed, based on the 23 conserved protein-coding genes of B. stricta and other 23 plant species, and the phylogenic relationship and evolution position of B. stricta were determined. The complete mt genome would be useful for further investigation of the genotype-by-environment interactions in mitochondria of Boechera.

Hotspot Selective Preference of the Chimeric Sequences Formed in Multiple Displacement Amplification

Multiple displacement amplification (MDA) is considered to be a conventional approach to comprehensive amplification from low input DNA. The chimeric reads generated in MDA lead to severe disruption in some studies, including those focusing on heterogeneity, structural variation, and genetic recombination. Meanwhile, the generation of by-products gives a new approach to gain insights into the reaction process of φ29 polymerase. Here, we analyzed 36.7 million chimeras and screened 196 billion chimeric hotspots in the human genome, as well as evaluating the hotspot selective preference of chimeras. No significant preference was captured in the distributions of chimeras and hotspots among chromosomes. Hotspots with overlaps for 12–13 nucleotides (nt) were most likely to be selected as templates in chimera generation. Meanwhile, a regularly selective preference was noticed in overlap GC content. The preferences in overlap length and GC content was shown to be pertinent to the sequence denaturation temperature, which pointed out the optimization direction for reducing chimeras. Distance preference between two segments of chimeras was 80–280 nt. The analysis is beneficial for reducing the chimeras in MDA, and the characterization of MDA chimeras is helpful in distinguishing MDA chimeras from chimeric sequences caused by disease.