Bi-PROF

Abstract

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10 (2)-10 (4) sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.

Epigenetic reprogramming during spermatogenesis and male factor infertility

“…Aberrations in sperm DNA methylation have also been shown to be associated with abnormalities in semen parameters (low sperm count, decreased volume of semen, reduced progressive motility, high percentage of immotile sperm lowered sperm vitality) of subfertile males that were trying to conceive for at least 10 years (Laqqan et al 2017a). The authors of this study identified that DNA methylation was consistently different at a small number of CpGs between proven fertile males and subfertile case subjects, using Infinium Human Methylation 450 K BeadChip arrays, validating previous findings using local deep bisulfite sequencing (Gries et al 2013). This study builds on earlier investigations, and recent studies by Nasri et al (2017) and Kobayashi et al (2017), showing that aberrant methylation, particularly at imprinted loci, is associated with poor-quality sperm (Marques et al 2004, Kobayashi et al 2007, Poplinski et al 2010, Pacheco et al 2011, Laurentino et al 2015.…”

Regional distribution of body fat in relation to DNA methylation within the LPL, ADIPOQ and PPARγ promoters in subcutaneous adipose tissue

“… 10 Experimental conduction was carried out as described previously 11 (details are given in Supplementary Table 2 ). SAT samples from three normal-weight and three obese participants were used (i) for selecting potentially informative CpG positions in the candidate genes using methylation data obtained from Illumina 450K BeadChip arrays and NGS-based bisulfite profiling focusing on absence of sequence polymorphism, high variability of methylation levels and association with BMI/gene expression (date not shown), and (ii) for validating SIRPH-based DNA methylation results with Bi-PROF. 12 The correlation between both methods was 0.97 ( P =0.005) for LPL -CG2, 0.87 ( P =0.024) for ADIPOQ -CG1 and 0.94 ( P =0.005) for PPARγ -CG1.…”

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males

“…According to the results of screening study, three CpG sites that have the greatest difference in methylation level between the case and control groups were subjected to validation using local deep bisulphite sequencing (Bi-PROF) (Gries et al, 2013) according to the manufacturer's instructions. In the validation study, 136 samples (independent samples) were used and distributed as follows: 72 samples as cases and 64 samples as controls.…”