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Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays

ABSTRACT: Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays

PROVIDER: PRJNA758354 | ENA |

REPOSITORIES: ENA

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Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays

Project description:Aim: Tandem bisulfite (BS) and oxidative bisulfite (oxBS) conversion on DNA followed by hybridization to Infinium HumanMethylation BeadChips allows nucleotide resolution of 5-hydroxymethylcytosine genome-wide. Here, the authors compared data quality acquired from BS-treated and oxBS-treated samples. Materials & methods: Raw BeadArray data from 417 pairs of samples across 12 independent datasets were included in the study. Probe call rates were compared between paired BS and oxBS treatments controlling for technical variables. Results: oxBS-treated samples had a significantly lower call-rate. Among technical variables, DNA-specific extraction kits performed better with higher call rates after oxBS conversion. Conclusion: The authors emphasize the importance of quality control during oxBS conversion to minimize information loss and recommend using a DNA-specific extraction kit for DNA extraction and an oxBSQC package for data preprocessing.

2022-01-14 | GSE182919 | GEO

Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays.

Project description:Aim: Tandem bisulfite (BS) and oxidative bisulfite (oxBS) conversion on DNA followed by hybridization to Infinium HumanMethylation BeadChips allows nucleotide resolution of 5-hydroxymethylcytosine genome-wide. Here, the authors compared data quality acquired from BS-treated and oxBS-treated samples. Materials & methods: Raw BeadArray data from 417 pairs of samples across 12 independent datasets were included in the study. Probe call rates were compared between paired BS and oxBS treatments controlling for technical variables. Results: oxBS-treated samples had a significantly lower call rate. Among technical variables, DNA-specific extraction kits performed better with higher call rates after oxBS conversion. Conclusion: The authors emphasize the importance of quality control during oxBS conversion to minimize information loss and recommend using a DNA-specific extraction kit for DNA extraction and an oxBSQC package for data preprocessing.

| S-EPMC8914583 | biostudies-literature

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

Project description:We performed a parallel analysis of commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se generates pronounced sequencing biases, and subsequent fragmentation and amplification steps lead to several-fold overrepresentation of these artefacts. Standard pre-bisulfite library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of methylation levels. We have integrated a bias diagnostic tool in the Bismark package and propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation.

2017-02-16 | GSE77961 | GEO

Bisulfite-free, nano-scale analysis of 5-hydroxymethylcytosine at single base resolution

Project description:High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples represents a continuous challenge. Here, we present CATCH-seq, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. CATCH-seq is based on selective 5hmC oxidation, labeling and subsequent C-to-T transition during PCR. Applications of CATCH-seq to nano-scale DNA samples reveal previously underappreciated non-CG 5hmCs in the hESC genome and base-resolution hydroxymethylome in human cell-free DNA.

2018-10-09 | GSE112048 | GEO

A novel approach uncovering the biological signal in Hi-C data affected by technical biases

Project description:The chromatin interaction assays, particularly Hi-C, enabled detailed studies of chromatin architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mediated by epigenetics mechanisms. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach HiConfidence eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and TADs. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in the histone acetylation pattern affect chromatin organization in Drosophila cells.

2023-02-05 | GSE200078 | GEO

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Project description:Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

2021-07-28 | GSE136940 | GEO

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

Project description:Nuclease-mediated depletion biases in ribosome footprint profiling libraries

| PRJNA613736 | ENA

OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA.

Project description:UnlabelledThe use of sodium bisulfite (BS) treatment followed by hybridization to an Illumina Infinium BeadChip (HumanMethylation450 and MethylationEPIC) is a common method for interrogating 5-methylcytosine (5mC) at single nucleotide resolution. However, standard treatment of DNA with BS does not allow disambiguation of 5mC from an additional cytosine modification, 5-hydroxymethylcytosine (5hmC). Recently, it has been demonstrated that paired BS and oxidative bisulfite (oxBS) treatment on the same sample followed by hybridization to an Infinium microarray permits the differentiation of 5hmC from 5mC. Nevertheless, estimation of 5hmC and 5mC from tandem-treated arrays has been shown to produce irregular estimates of cytosine modifications.ResultsWe present a novel method using maximum likelihood estimation to accurately estimate the parameters of unmethylated cytosine (5C), 5mC and 5hmC from Infinium microarray data given the signal intensities from the oxBS and BS replicates.Availability and implementationOxyBS is an R package available on CRAN.ContactAndres.Houseman@oregonstate.eduSupplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC4978924 | biostudies-literature

Biases in ethanol-based DNA metabarcoding

Project description:Modelling technical and biological biases in macroinvertebrate community assessment from bulk preservative using multiple metabarcoding markers

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The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5M-bM-^@M-2 uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5M-bM-^@M-^Y end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis Immunoprecipitated small RNA were purified from Bmn4 cells for preparation of high-throughput sequencing libraries.

2014-07-01 | E-GEOD-55451 | biostudies-arrayexpress

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