Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA271137 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1734730.fastq.gz | Fastqsanger.gz | |||
| SRR1734740.fastq.gz | Fastqsanger.gz | |||
| SRR1734731.fastq.gz | Fastqsanger.gz | |||
| SRR1734741.fastq.gz | Fastqsanger.gz | |||
| SRR1734732.fastq.gz | Fastqsanger.gz |
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Project description:ChIP-seq experiments are standard experimental procedure for interrogating epigenetic states and protein-DNA interactions. Sequencing experiments are often designed according to the trade-off between the need to obtain maximum sequencing coverage limited funds. Multiplexing samples is a common approach to minimize cost and maximize information yield. We therefore performed an extensive ChiP-seq multiplexing study to gain a better understanding of the effect of multiplexing on the resulting peak detection and genomic annotation and to provide solid guidelines for multiplexing ChIP-seq studies. For a well characterized antibody, our results indicate that multiplexing to ~20M reads (roughly 8 samples per sequencing lane) is sufficient to capture most of the biological signal. Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed an analysis of peak detection to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million reads each and still detect over 90% of all peaks found when using a full lane for sample. Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.
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Project description:Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly double the multiplexing capability of the TMTpro reagent set to 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak co-elution, which can compromise the accuracy of reporter ion-based quantification. To counteract the impact of the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of non-deuterium and deuterium-containing channels into distinct sub-plexes during normalization procedures with reassembly through a common bridge channel. This multiplexing strategy of “design independent sub-plexes but acquire together” was used to compare protein expression differences between human cell lines and in a cysteine-profiling (e.g., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.
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Project description:The proposed method uses enriched de novo motifs on the peak sequences of Ago2-CLIP to determine miRNA-RNA heteroduplex for either canonical or non-canonical miRNA-binding sites. miRNA-RNA heteroduplex are ranked based on a duplex score. Since the quality of the peak detection is of pivotal importance for the final output of the proposed method, we also provide an illustrative comparative evaluation of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, for four publicly available Ago2-datasets and one previously unpublished in-house Ago2-dataset in stem cells. The experimental validation was carried out on the in-houseAgo2 dataset by gene reporter assay.
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Project description:ChIP-Seq for H3K27 trimethylation was performed for two HPV-positive and two HPV-negative squamous cell carcinoma cell lines. The data served two purposes. First, the data were used as an example implementation of our novel ChIP-Seq Peak Prioritization pipeline, PePr. We have developed the PePr pipeline, a ChIP-Seq Peak Prioritization pipeline that accounts for the variation among replicates and peak location relative to a gene. We show, using a transcription factor dataset (which exhibited small variation among samples), that PePr performs favorably compared to commonly used peak callers and that it achieves balanced sensitivity and specificity. We also show, using histone modification data (which exhibited larger variation among samples), that PePr can improve the detection of differential H3K27me3 regions compared with a common current approach. Using data from ChIP-Seq and gene expression experiments performed in parallel on the same samples, we show that the incorporation of functional annotations can improve the prioritization of functional sites. Secondly, the data were used to assess real differences in the genome-wide H3K27me3 profiles between HPV-positive and HPV-negative carcinoma cell lines. Careful analysis and integration of the data with DNA methylation and gene expression data performed on the same cell lines demonstrated striking differences exist.
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Project description:Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However multiplexing involves a tradeoff between increased number of sequenced samples and reduced number of reads per sample (i.e. lower coverage). To assess the effect of different depths of coverage due to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing 1, 3, 6, 9, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression greater than 1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the Log2 number of reads aligning to microRNA loci (R=0.96). However, most additional microRNAs detected in samples with greater sequencing coverage were in the range of expression which had lower fold change reproducibility. These findings elucidate the tradeoff between increasing the number of samples in a multiplex with decreasing coverage and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.
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