RRBS Analysis Report for in2514
This report includes summaries of data quality, data processing, and snapshots of the results for your DNA methylation study. This report should assist you to get a general picture of the study and to spot any irregularities in the sample or data. Additional data is provided with Full Bioinformatic Analysis package.
General Statistics
Showing 8/16 rows and 6/23 columns.Sample Name | # Reads | % Uniquely Aligned | % BS Conversion (Non-CpG) | % BS Conversion (Spike-in) | Uniq. CpG | Avg. CpG Coverage |
---|---|---|---|---|---|---|
FFPE_NAT_Lung_1 | 43537529 | 59.4% | 98.8% | 99.4% | 6935178 | 12X |
FFPE_NAT_Lung_2 | 37815108 | 59.2% | 98.6% | 99.3% | 6753440 | 11X |
FFPE_Tumor_Lung_1 | 26540656 | 58.5% | 98.7% | 99.3% | 6248673 | 8X |
FFPE_Tumor_Lung_2 | 27606850 | 58.3% | 98.5% | 99.2% | 6325213 | 9X |
NAT_Liver_1 | 9961574 | 63.6% | 98.9% | 99.0% | 6866793 | 4X |
NAT_Liver_2 | 13858872 | 63.9% | 99.0% | 99.3% | 7110409 | 6X |
Tumor_Liver_1 | 32750371 | 63.0% | 99.0% | 99.1% | 9013360 | 10X |
Tumor_Liver_2 | 12187163 | 62.6% | 98.9% | 99.1% | 7247017 | 5X |
Cutadapt
Cutadapt is a tool to find and remove adapter sequences, primers, poly-Atails and other types of unwanted sequence from your high-throughput sequencing reads.
Trimmed Sequence Lengths
This plot shows the number of reads with certain lengths of adapter trimmed.
Obs/Exp shows the raw counts divided by the number expected due to sequencing errors. A defined peak may be related to adapter length.
See the cutadapt documentation for more information on how these numbers are generated.
Filtered Reads
This plot shows the number of reads (single-end) or read pairs (paired-end) passing filters.
FastQC (trimmed)
FastQC is a quality control tool for high throughput sequence data, written by Simon Andrews at the Babraham Institute in Cambridge.
This section of the report shows FastQC results after adapter and quality trimming.Sequence Quality Histograms
The mean quality value across each base position in the read.
To enable multiple samples to be plotted on the same graph, only the mean quality scores are plotted (unlike the box plots seen in FastQC reports).
Taken from the FastQC help:
The y-axis on the graph shows the quality scores. The higher the score, the better the base call. The background of the graph divides the y axis into very good quality calls (green), calls of reasonable quality (orange), and calls of poor quality (red). The quality of calls on most platforms will degrade as the run progresses, so it is common to see base calls falling into the orange area towards the end of a read.
Per Sequence Quality Scores
The number of reads with average quality scores. Shows if a subset of reads has poor quality.
From the FastQC help:
The per sequence quality score report allows you to see if a subset of your sequences have universally low quality values. It is often the case that a subset of sequences will have universally poor quality, however these should represent only a small percentage of the total sequences.
Per Base Sequence Content
The proportion of each base position for which each of the four normal DNA bases has been called.
To enable multiple samples to be shown in a single plot, the base composition data is shown as a heatmap. The colours represent the balance between the four bases: an even distribution should give an even muddy brown colour. Hover over the plot to see the percentage of the four bases under the cursor.
To see the data as a line plot, as in the original FastQC graph, click on a sample track.
From the FastQC help:
Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called.
In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other. The relative amount of each base should reflect the overall amount of these bases in your genome, but in any case they should not be hugely imbalanced from each other.
It's worth noting that some types of library will always produce biased sequence composition, normally at the start of the read. Libraries produced by priming using random hexamers (including nearly all RNA-Seq libraries) and those which were fragmented using transposases inherit an intrinsic bias in the positions at which reads start. This bias does not concern an absolute sequence, but instead provides enrichement of a number of different K-mers at the 5' end of the reads. Whilst this is a true technical bias, it isn't something which can be corrected by trimming and in most cases doesn't seem to adversely affect the downstream analysis.
Rollover for sample name
Per Sequence GC Content
The average GC content of reads. Normal random library typically have a roughly normal distribution of GC content.
From the FastQC help:
This module measures the GC content across the whole length of each sequence in a file and compares it to a modelled normal distribution of GC content.
In a normal random library you would expect to see a roughly normal distribution of GC content where the central peak corresponds to the overall GC content of the underlying genome. Since we don't know the the GC content of the genome the modal GC content is calculated from the observed data and used to build a reference distribution.
An unusually shaped distribution could indicate a contaminated library or some other kinds of biased subset. A normal distribution which is shifted indicates some systematic bias which is independent of base position. If there is a systematic bias which creates a shifted normal distribution then this won't be flagged as an error by the module since it doesn't know what your genome's GC content should be.
Overrepresented sequences
The total amount of overrepresented sequences found in each library.
FastQC calculates and lists overrepresented sequences in FastQ files. It would not be possible to show this for all samples in a MultiQC report, so instead this plot shows the number of sequences categorized as over represented.
Sometimes, a single sequence may account for a large number of reads in a dataset. To show this, the bars are split into two: the first shows the overrepresented reads that come from the single most common sequence. The second shows the total count from all remaining overrepresented sequences.
From the FastQC Help:
A normal high-throughput library will contain a diverse set of sequences, with no individual sequence making up a tiny fraction of the whole. Finding that a single sequence is very overrepresented in the set either means that it is highly biologically significant, or indicates that the library is contaminated, or not as diverse as you expected.
FastQC lists all of the sequences which make up more than 0.1% of the total. To conserve memory only sequences which appear in the first 100,000 sequences are tracked to the end of the file. It is therefore possible that a sequence which is overrepresented but doesn't appear at the start of the file for some reason could be missed by this module.
Adapter Content
The cumulative percentage count of the proportion of your library which has seen each of the adapter sequences at each position.
Note that only samples with ≥ 0.1% adapter contamination are shown.
There may be several lines per sample, as one is shown for each adapter detected in the file.
From the FastQC Help:
The plot shows a cumulative percentage count of the proportion of your library which has seen each of the adapter sequences at each position. Once a sequence has been seen in a read it is counted as being present right through to the end of the read so the percentages you see will only increase as the read length goes on.
Bismark
Bismark is a tool to map bisulfite converted sequence reads and determine cytosine methylation states.
Alignment Rates
Strand Alignment
M-Bias
This plot shows the average percentage methylation and coverage across reads. See the bismark user guide for more information on how these numbers are generated.
Insert Size
This section of the report has a plot, generated by Picard, that shows the number of reads at a given insert size. Reads with different orientation are summed.
CpG Coverage
CpG Coverage shows the distribution of the detected CpG coverage in each library.
Genomic Region Coverage
This section presents the percentage of a certain type of genomic functional region that is covered in each RRBS library, categorized by coverage. The genomic functional region of interest includes CpG islands, gene promoters, and gene bodies. These regions are derived from public annotation files of the reference assemblies.
Promoter
Promoter Coverage shows the distribution of the number of individual CpG methylation measurements in promoters genome wide.
Gene Body
Gene Body Coverage shows the distribution of the number of individual CpG methylation measurements in gene bodies genome wide.
CpG Island
CpG Island Coverage shows the distribution of the number of individual CpG methylation measurements in GpG islands genome wide.
Bismark (spike-in)
Bismark is a tool to map bisulfite converted sequence reads and determine cytosine methylation states.
This section of the report shows Bismark results for spike-in reads.Strand Alignment
Samtools (spike-in)
Samtools is a suite of programs for interacting with high-throughput sequencing data.
This section of the report shows Samtools results for spike-in reads.Mapped reads per contig
The samtools idxstats
tool counts the number of mapped reads per chromosome / contig. Chromosomes with < 0.1% of the total aligned reads are omitted from this plot.
MethylDackel (spike-in)
MethylDackel is a per-base methylation metrics extraction tool for BAM file with BS-seq alignments.
This section of the report shows the average methylation ratio per contig for spike-in reads.Correlation Scatter Plot
This plot shows the correlation trend between observed and expected values.
Correlation Table
This table shows the Pearson's Correlation Coefficient for each sample.
Sample Name | Correlation Coefficient |
---|---|
FFPE_NAT_Lung_1 | 0.978 |
FFPE_NAT_Lung_2 | 0.978 |
FFPE_Tumor_Lung_1 | 0.983 |
FFPE_Tumor_Lung_2 | 0.979 |
NAT_Liver_1 | 0.977 |
NAT_Liver_2 | 0.974 |
Tumor_Liver_1 | 0.972 |
Tumor_Liver_2 | 0.979 |
Download
This section contains links to download trimmed fastq files, data and/or images generated by various bioinformatics tools. To download individual files, click on the corresponding links. The links will expire in 90 days.
Sample Name | Trimmed Fastq R1 | Trimmed Fastq R2 | Alignment | DNA Methylation Calling | Browser Track | Methylation Distribution |
---|---|---|---|---|---|---|
FFPE_NAT_Lung_1 | Download | Download | Download | Download | Download | Download |
FFPE_NAT_Lung_2 | Download | Download | Download | Download | Download | Download |
FFPE_Tumor_Lung_1 | Download | Download | Download | Download | Download | Download |
FFPE_Tumor_Lung_2 | Download | Download | Download | Download | Download | Download |
NAT_Liver_1 | Download | Download | Download | Download | Download | Download |
NAT_Liver_2 | Download | Download | Download | Download | Download | Download |
Tumor_Liver_1 | Download | Download | Download | Download | Download | Download |
Tumor_Liver_2 | Download | Download | Download | Download | Download | Download |
Instructions to download all files
cd ~/Download
and Enter. If the code is downloaded to a different location, change ~/Download
to the path to that folder.bash download_links.ps1
(Mac/Linux) or Powershell.exe -ExecutionPolicy Bypass -File .\download_links.ps1
(Windows) and Enter.Summary
- Library Type
- RRBS
- Directionality
- Non-directional
- Genome
- hg19
- Trimming
- 5'R1: 0bp / 5'R2: 0bp / Illumina adapter / RRBS: On
Software Versions
Versions are collected at run time from the software output.
- fastqc
- 0.11.9
- trimgalore
- 0.6.4_dev
- bismark
- 0.22.3
- picard
- 2.23.8
- samtools
- 1.9
- methyldackel
- 0.5.0
- nextflow
- 20.07.1
- pipeline
- dev