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

<div><p>Background</p><p>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. <i>coli</i> genome.</p><p>Method</p><p>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.</p><p>Results/Conclusion</p><p>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.</p><p>Significance</p><p>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.</p></div

The curve of the 18 subsamples about the relationship between the average insertion size and the ratio of the insertion chimeras to the pair-end chimeras.

<p>The curve of the 18 subsamples about the relationship between the average insertion size and the ratio of the insertion chimeras to the pair-end chimeras.</p

The visualization of the chimeras with the read parts located on the reverse strands (1) or on the same strands (2).

<p>The visualization of the chimeras with the read parts located on the reverse strands (1) or on the same strands (2).</p

The quantity statistics of the pair-end chimeras with two parts on the reverse strands or on the same strands.

<p>The quantity statistics of the pair-end chimeras with two parts on the reverse strands or on the same strands.</p

The characteristics of the sequence data of the varied-level chimeras.

<p>(1a) (1b) 1-level chimera; (2a) (2b) 2-level chimera; (3) 3-level chimera.</p

The abundance distribution graph of the physical genomic distance of the two parts of the reads and the length of the overlap segments.

<p>Sample SRX247249 was illustrated in Fig 4–1, and Sample SRX252522 was illustrated in Fig 4–2.</p

The amount of varied-level chimeras and the total proportion of chimeras.

<p>The amount of varied-level chimeras and the total proportion of chimeras.</p

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