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Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. The bioinformatic framework successfully maps and clusters DNA sequences The experimental protocols described in this study Figs 1 and 2 aim to improve the existing techniques and provide the means to investigate the effects of oxidative damage to the genetic content of sperm cells.
Both files arranged their data into a BED format to allow visual inspection of the coverage and counts within each region in a genomic browser such as the UCSC Genome Browser [ 47 ]. Additionally, the concentration and the duration of the exposure to DTT was reduced to a level that minimised the induction of lesions but sufficient to break disulphide bonds and relax the overall chromatin structure [ 62 — 64 ].
March 14, 2018; Published: Although method 2 Sonication after lysis did not generate as much oxidatively damage DNA than method 4 MNase after lysis , it nonetheless increased the levels of oxidation relatively to controls. Atmos Chem Phys.
Although past studies have suggested that MNase digest the DNA preferentially at specific genomic locations [ 57 — 59 ], this apparent specificity has been linked to chromatin accessibility.
A small number of studies have utilised modified chromatin immunoprecipitation techniques ChIP [ 25 — 27 ] in conjunction with high-throughput sequencing [ 28 , 29 ] in an attempt to identify genomic sites vulnerable to oxidative attack and subsequently correlate this information with chromatin structure and the arrangement of DNA packaging inside the nucleus of mature spermatozoa [ 30 — 34 ].
In this study, we showed that the combination of MNase digestion to generate DNA fragments and removal of oxidising and reducing compounds from cell lysis and DNA extraction processes improved significantly the experimental yield while inflicting the least amount of oxidative damage to DNA. An alternative method where MNase digestion took place after cell lysis and the isolation of DNA was developed and tested but the results demonstrated a comparative increase in the amount of oxidative damage.
Mock sequence data arranged into a single file in FASTA format was mapped against the human reference genome using Bowtie [ 43 ] Fig 3. At this stage of the analysis it was necessary to sort and group mapped sequences to determine the boundaries of the regions of containing oxidised DNA within them.
Half the sequences were chosen consecutively from two 200 bp region, separated by 100 bp, so sequences overlapped for 5 bp at each end with surrounding sequences. Mickey B, Howard J. The modified protocols developed to better investigate the impact of oxidative stress on human sperm DNA Fig 1 were successfully adapted from existing methods for DNA extraction from spermatozoa [ 4 , 28 — 30 , 35 — 37 ] by completely removing or minimising the presence of oxidising chemical agents not strictly necessary for DNA extraction and fragmentation.
Due to the unstable nature of these compounds and their tendency to facilitate oxidation of dissolved oxygen by electron donation during redox activity [ 67 , 69 , 70 ], ROS becomes more abundant and starts to inflict oxidative damage to the DNA in the cells.
At this stage of the bioinformatic framework, sequencing data could no longer be analysed separately and individual sample results needed to be taken in context with results from controls. Human Donor spermatozoa samples, isolated via Percoll density gradient centrifugation and cleaned of leucocytes with Dynabeads, were pooled together in a single sample 0.
Successfully mapped sequences were then sorted by chromosome and by start position by another Perl algorithm that separated aligned sequences from those with multiple alignments or too ambiguous to align to a known position. Sperm-derived histones contribute to zygotic chromatin in humans. Statistical significance of the variance in collateral oxidative damage inflicted between methods was determined by performing a Kruskal-Wallis one-way analysis of variance and the post-hoc multiple comparison test using the kruskalmc function in the pgirmess package in R [ 49 ].
Consequently, samples of spermatozoa from healthy normozoospermic human donors Fig 2.
We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.
We propose that prolonged exposure of naked DNA to oxidative agents during the multiple rounds of phenol-chloroform extractions and losses inflicted over the course of successive washing steps provide a viable explanation for the observed reduction in yield and increased amount of oxidative lesion at the end of this experimental method.
With this goal in mind, compounds with highly reactive hydroxyl radicals -OH , superoxide radicals O 2 - and oxidation catalysers were completely removed, or significantly reduced, from the existing protocols to prevent the generation of ROS and free radicals capable of inducing oxidative lesions. Thus, method 3 MNase after cell lysis continued to be surpassed in terms of efficiency by both methods 2 sonication and 4 MNase before cell lysis Fig 4B. Sperm chromatin structure assay: This option was coded into the program to allow for instances where close sequences were less than a sequence away from each other, an occurrence predicted to occur in real datasets due to sequencing errors or ambiguous mapping.