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        Download the raw data used to create the plots in this report below:

        Note that additional data was saved in multiqc_data when this report was generated.


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        If you use plots from MultiQC in a publication or presentation, please cite:

        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

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        About MultiQC

        This report was generated using MultiQC, version 1.7

        You can see a YouTube video describing how to use MultiQC reports here: https://youtu.be/qPbIlO_KWN0

        For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

        You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

        MultiQC is published in Bioinformatics:

        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

        A modular tool to aggregate results from bioinformatics analyses across many samples into a single report.

        This report has been generated by the nf-core/rnaseq analysis pipeline. For information about how to interpret these results, please see the documentation.

        Report generated on 2020-10-27, 17:42 based on data in: /home/srlab/Documents/Shelly/work/58/eebd6dfe27857f1cf43b7f79ad8f95


        General Statistics

        Showing 8/8 rows and 11/14 columns.
        Sample Name% rRNAdupInt5'-3' biasM Aligned% AssignedM Assigned% AlignedM Aligned% Trimmed% GCM Seqs
        SRR4238351
        0.00%
        0.34
        0.0
        59.0%
        0.0
        99.5%
        0.0
        0.3%
        44%
        0.0
        SRR4238351_subsamp
        0.00%
        SRR4238355
        0.01%
        0.78
        0.0
        54.1%
        0.0
        99.8%
        0.0
        0.3%
        44%
        0.0
        SRR4238355_subsamp
        0.00%
        SRR4238359
        0.00%
        1.50
        0.0
        57.1%
        0.0
        99.9%
        0.0
        0.3%
        45%
        0.0
        SRR4238359_subsamp
        0.00%
        SRR4238379
        0.00%
        0.77
        0.0
        56.6%
        0.0
        99.6%
        0.0
        0.3%
        44%
        0.0
        SRR4238379_subsamp
        0.00%

        Biotype Counts

        Biotype Counts shows reads overlapping genomic features of different biotypes, counted by featureCounts.

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        edgeR: Sample Similarity

        edgeR: Sample Similarity is generated from normalised gene counts through edgeR. Pearson's correlation between log2 normalised CPM values are then calculated and clustered.

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        MDS Plot

        MDS Plot show relatedness between samples in a project. These values are calculated using edgeR in the edgeR_heatmap_MDS.r script.

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        DupRadar

        DupRadar provides duplication rate quality control for RNA-Seq datasets. Highly expressed genes can be expected to have a lot of duplicate reads, but high numbers of duplicates at low read counts can indicate low library complexity with technical duplication. This plot shows the general linear models - a summary of the gene duplication distributions.

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        QualiMap

        QualiMap is a platform-independent application to facilitate the quality control of alignment sequencing data and its derivatives like feature counts.

        Genomic origin of reads

        Classification of mapped reads as originating in exonic, intronic or intergenic regions. These can be displayed as either the number or percentage of mapped reads.

        There are currently three main approaches to map reads to transcripts in an RNA-seq experiment: mapping reads to a reference genome to identify expressed transcripts that are annotated (and discover those that are unknown), mapping reads to a reference transcriptome, and de novo assembly of transcript sequences (Conesa et al. 2016).

        For RNA-seq QC analysis, QualiMap can be used to assess alignments produced by the first of these approaches. For input, it requires a GTF annotation file along with a reference genome, which can be used to reconstruct the exon structure of known transcripts. This allows mapped reads to be grouped by whether they originate in an exonic region (for QualiMap, this may include 5′ and 3′ UTR regions as well as protein-coding exons), an intron, or an intergenic region (see the Qualimap 2 documentation).

        The inferred genomic origins of RNA-seq reads are presented here as a bar graph showing either the number or percentage of mapped reads in each read dataset that have been assigned to each type of genomic region. This graph can be used to assess the proportion of useful reads in an RNA-seq experiment. That proportion can be reduced by the presence of intron sequences, especially if depletion of ribosomal RNA was used during sample preparation (Sims et al. 2014). It can also be reduced by off-target transcripts, which are detected in greater numbers at the sequencing depths needed to detect poorly-expressed transcripts (Tarazona et al. 2011).

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        Gene Coverage Profile

        Mean distribution of coverage depth across the length of all mapped transcripts.

        There are currently three main approaches to map reads to transcripts in an RNA-seq experiment: mapping reads to a reference genome to identify expressed transcripts that are annotated (and discover those that are unknown), mapping reads to a reference transcriptome, and de novo assembly of transcript sequences (Conesa et al. 2016).

        For RNA-seq QC analysis, QualiMap can be used to assess alignments produced by the first of these approaches. For input, it requires a GTF annotation file along with a reference genome, which can be used to reconstruct the exon structure of known transcripts. QualiMap uses this information to calculate the depth of coverage along the length of each annotated transcript. For a set of reads mapped to a transcript, the depth of coverage at a given base position is the number of high-quality reads that map to the transcript at that position (Sims et al. 2014).

        QualiMap calculates coverage depth at every base position of each annotated transcript. To enable meaningful comparison between transcripts, base positions are rescaled to relative positions expressed as percentage distance along each transcript (0%, 1%, …, 99%). For the set of transcripts with at least one mapped read, QualiMap plots the cumulative mapped-read depth (y-axis) at each relative transcript position (x-axis). This plot shows the gene coverage profile across all mapped transcripts for each read dataset. It provides a visual way to assess positional biases, such as an accumulation of mapped reads at the 3′ end of transcripts, which may indicate poor RNA quality in the original sample (Conesa et al. 2016).

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        Preseq

        Preseq estimates the complexity of a library, showing how many additional unique reads are sequenced for increasing total read count. A shallow curve indicates complexity saturation. The dashed line shows a perfectly complex library where total reads = unique reads.

        Complexity curve

        Note that the x axis is trimmed at the point where all the datasets show 80% of their maximum y-value, to avoid ridiculous scales.

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        RSeQC

        RSeQC package provides a number of useful modules that can comprehensively evaluate high throughput RNA-seq data.

        Read Distribution

        Read Distribution calculates how mapped reads are distributed over genome features.

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        Read Duplication

        read_duplication.py calculates how many alignment positions have a certain number of exact duplicates. Note - plot truncated at 500 occurrences and binned.

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        Junction Saturation

        Junction Saturation counts the number of known splicing junctions that are observed in each dataset. If sequencing depth is sufficient, all (annotated) splice junctions should be rediscovered, resulting in a curve that reaches a plateau. Missing low abundance splice junctions can affect downstream analysis.

        Click a line to see the data side by side (as in the original RSeQC plot).

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        Infer experiment

        Infer experiment counts the percentage of reads and read pairs that match the strandedness of overlapping transcripts. It can be used to infer whether RNA-seq library preps are stranded (sense or antisense).

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        featureCounts

        Subread featureCounts is a highly efficient general-purpose read summarization program that counts mapped reads for genomic features such as genes, exons, promoter, gene bodies, genomic bins and chromosomal locations.

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        STAR

        STAR is an ultrafast universal RNA-seq aligner.

        Alignment Scores

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        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.

        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.

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        FastQC

        FastQC is a quality control tool for high throughput sequence data, written by Simon Andrews at the Babraham Institute in Cambridge.

        Sequence Counts

        Sequence counts for each sample. Duplicate read counts are an estimate only.

        This plot show the total number of reads, broken down into unique and duplicate if possible (only more recent versions of FastQC give duplicate info).

        You can read more about duplicate calculation in the FastQC documentation. A small part has been copied here for convenience:

        Only sequences which first appear in the first 100,000 sequences in each file are analysed. This should be enough to get a good impression for the duplication levels in the whole file. Each sequence is tracked to the end of the file to give a representative count of the overall duplication level.

        The duplication detection requires an exact sequence match over the whole length of the sequence. Any reads over 75bp in length are truncated to 50bp for this analysis.

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        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.

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        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.

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        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.

        Click a sample row to see a line plot for that dataset.
        Rollover for sample name
        Position: -
        %T: -
        %C: -
        %A: -
        %G: -

        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.

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        Per Base N Content

        The percentage of base calls at each position for which an N was called.

        From the FastQC help:

        If a sequencer is unable to make a base call with sufficient confidence then it will normally substitute an N rather than a conventional base call. This graph shows the percentage of base calls at each position for which an N was called.

        It's not unusual to see a very low proportion of Ns appearing in a sequence, especially nearer the end of a sequence. However, if this proportion rises above a few percent it suggests that the analysis pipeline was unable to interpret the data well enough to make valid base calls.

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        Sequence Length Distribution

        The distribution of fragment sizes (read lengths) found. See the FastQC help

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        Sequence Duplication Levels

        The relative level of duplication found for every sequence.

        From the FastQC Help:

        In a diverse library most sequences will occur only once in the final set. A low level of duplication may indicate a very high level of coverage of the target sequence, but a high level of duplication is more likely to indicate some kind of enrichment bias (eg PCR over amplification). This graph shows the degree of duplication for every sequence in a library: the relative number of sequences with different degrees of duplication.

        Only sequences which first appear in the first 100,000 sequences in each file are analysed. This should be enough to get a good impression for the duplication levels in the whole file. Each sequence is tracked to the end of the file to give a representative count of the overall duplication level.

        The duplication detection requires an exact sequence match over the whole length of the sequence. Any reads over 75bp in length are truncated to 50bp for this analysis.

        In a properly diverse library most sequences should fall into the far left of the plot in both the red and blue lines. A general level of enrichment, indicating broad oversequencing in the library will tend to flatten the lines, lowering the low end and generally raising other categories. More specific enrichments of subsets, or the presence of low complexity contaminants will tend to produce spikes towards the right of the plot.

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        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.

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        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.

        No samples found with any adapter contamination > 0.1%

        nf-core/rnaseq Software Versions

        nf-core/rnaseq Software Versions are collected at run time from the software output.

        nf-core/rnaseq
        v1.4.2
        Nextflow
        v20.07.1
        FastQC
        v0.11.8
        Cutadapt
        v2.5
        Trim Galore!
        v0.6.4
        SortMeRNA
        v2.1b
        STAR
        vSTAR_2.6.1d
        HISAT2
        v2.1.0
        Picard MarkDuplicates
        v2.21.1
        Samtools
        v1.9
        featureCounts
        v1.6.4
        Salmon
        v0.14.1
        StringTie
        v2.0
        Preseq
        v2.0.3
        deepTools
        v3.3.1
        RSeQC
        v3.0.1
        dupRadar
        v1.14.0
        edgeR
        v3.26.5
        Qualimap
        v.2.2.2-dev
        MultiQC
        v1.7

        nf-core/rnaseq Workflow Summary

        nf-core/rnaseq Workflow Summary - this information is collected when the pipeline is started.

        Pipeline Release
        master
        Run Name
        nasty_legentil
        Reads
        data/*{1,2}.fastq.gz
        Data Type
        Single-End
        Strandedness
        None
        Trimming
        5'R1: 0 / 5'R2: 0 / 3'R1: 0 / 3'R2: 0 / NextSeq Trim: 0
        Aligner
        STAR
        Fasta Ref
        https://github.com/nf-core/test-datasets/raw/rnaseq/reference/genome.fa
        GTF Annotation
        https://github.com/nf-core/test-datasets/raw/rnaseq/reference/genes.gtf
        GFF3 Annotation
        https://github.com/nf-core/test-datasets/raw/rnaseq/reference/genes.gff
        Remove Ribosomal RNA
        N/A
        Biotype GTF field
        gene_biotype
        Save prefs
        Ref Genome: No / Trimmed FastQ: No / Alignment intermediates: No
        Max Resources
        6 GB memory, 2 cpus, 2d time per job
        Container
        docker - nfcore/rnaseq:1.4.2
        Output dir
        ./results
        Launch dir
        /home/srlab/Documents/Shelly
        Working dir
        /home/srlab/Documents/Shelly/work
        Script dir
        /home/srlab/.nextflow/assets/nf-core/rnaseq
        User
        srlab
        Config Profile
        test,docker
        Config Description
        Minimal test dataset to check pipeline function