This interactive report provides an overview of m6A-eCLIP results, accessible at the different tabs. For each table and plot, the button can be clicked to get more information. The menus in the top right corner of each plot can be used to modify the plots (zoom, hide samples, and save a copy of the plot). This report is optimized for viewing on a desktop and plots have been tested on several browsers but in some cases they may not render correctly, please use an alternate browser if plots do not load.
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Experiment Summary
This report summarizes a m6A-eCLIP experiment comparing HEK293 to K562 cells. Samples were run using Eclipsebio's proprietary m6A-eCLIP technology to identify m6A sites. Full methods details are available upon request.
Quality Control Metrics
The following table shows important quality control metrics for each sample. Two libraries are made for each sample, one containing sequences immunoprecipitated with the antibody of interest (IP), and one RNA-seq background control (input). For each library, one row is included that lists the metrics for each library. The identity of the library is included in the "IP or input" column. The following columns describe the processing of the libraries:
- Initial reads: the number of sequenced reads for each library.
- % Pass trim: reads first underwent adapter trimming to remove both adapters and sequences shorter than 18 nucleotides. The percentage of sequenced reads that passed this trimming step is listed in this column.
- % Repetitive elements: trimmed reads are aligned to Eclipsebio's custom database of repetitive elements. This database includes rRNAs, tRNAs, snoRNAs, and other features. The percentage of trimmed reads that were filtered is listed in the column.
- % Uniquely aligned to the genome: the remaining reads from the repetitive element filtering were aligned to the genome, and the percent of those reads that map uniquely to the genome is listed in this column.
- % PCR duplicates: PCR duplicates were defined as uniquely mapped reads that map to the same read coordinates and have an identical unique molecular identifier (UMI). The percentage of mapped reads that were identified as PCR duplicates is included in this column.
- Final reads: this column lists the number of uniquely mapped, deduplicated reads that were used for this analysis.
- Clusters: Clusters were defined as regions on genes within the IP sample that contain an enrichment of read coverage. These clusters were identified by the peak called CLIPper. The number of clusters called in each IP sample is listed in this column. Clusters are not called for input samples.
- Input-normalized peaks: For every cluster called, an IP vs. input log2 fold change and associated p-value were calculated using the Yates' Chi-square test or Fisher Exact test if read counts were < 5. Peaks were defined as clusters with log2 fold enrichment > 3 and p-value < 0.001. The number of peaks identified for each IP sample is listed in this column. Peaks are not called for input samples.
- Single nucleotide sites: Single nucleotide sites were identified using the tool PureCLIP. Called sites are enriched in the IP sample over the corresponding input control sample.
- Filtered m6A sites: Single nucleotide sites filtered for the following criteria: score ≥ 10, on an "A" nucleotide, and intersects with a significantly enriched m6A peak.
| Sample | IP or Input | Initial reads | % Pass trim | % Repetitive elements | % Uniquely aligned to genome | % PCR duplicates | Final reads | Clusters | Input-normalized peaks | Single nucleotide sites | Filtered m6A sites |
|---|---|---|---|---|---|---|---|---|---|---|---|
| K562_Rep1_IP | IP | 56,701,623 | 95.96% | 4.15% | 77.80% | 38.07% | 25,127,627 | 280,969 | 9,377 | 245,164 | 2,324 |
| K562_Rep2_IP | IP | 54,198,813 | 96.96% | 4.29% | 77.46% | 38.19% | 24,078,213 | 280,957 | 9,362 | 224,464 | 2,244 |
| HEK293_Rep1_IP | IP | 48,281,489 | 98.37% | 4.75% | 84.13% | 33.90% | 25,154,707 | 294,825 | 21,566 | 195,867 | 9,891 |
| HEK293_Rep2_IP | IP | 50,110,964 | 98.59% | 4.74% | 83.71% | 37.10% | 24,777,770 | 294,804 | 19,987 | 185,700 | 8,765 |
| K562_Rep1_Input | Input | 44,052,629 | 99.45% | 7.02% | 78.82% | 18.57% | 26,145,088 | ||||
| K562_Rep2_Input | Input | 48,007,021 | 99.50% | 7.27% | 78.96% | 19.64% | 28,107,547 | ||||
| HEK293_Rep1_Input | Input | 54,785,657 | 99.84% | 9.44% | 85.46% | 22.61% | 32,757,241 | ||||
| HEK293_Rep2_Input | Input | 56,595,373 | 99.86% | 9.37% | 85.28% | 24.02% | 33,191,998 |
This tab shows the m6A peaks in each sample meeting cutoffs of IP vs. input log2 fold enrichment ≥ 3 and p-value ≤ 0.001, as well as m6A peaks found to be reproducible between replicates (if applicable).
Peak Counts
Clusters are found using the peak calling tool CLIPper. The clusters identified in the IP sample are then normalized against a paired input sample. A peak is defined as a cluster with log2 fold enrichment ≥ 3 and p-value ≤ 0.001. Reproducible peaks are identified using Eclipsebio's eCV analysis. The bar plot below shows the number of significantly enriched peaks detected in each sample, as well as the number of significantly enriched reproducible peaks for each set of replicates. Hover over each bars to see the number of peaks in each set. All plots in this report are interactive, meaning you can zoom in/out, pan to a certain location, and select elements from the legend to remove. Plots can also be downloaded as a separate file. Hover over the top right corner of the plot to see the interactive options.