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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 1.8

        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.

        Report generated on 2020-04-26, 14:32 based on data in: /home/sam/analyses/20200426_olur_fastqc_quantseq


        General Statistics

        Showing 146/146 rows and 3/5 columns.
        Sample Name% Dups% GCM Seqs
        137_S63_L001_R1_001
        45.9%
        35%
        6.4
        139_S54_L001_R1_001
        34.8%
        35%
        2.3
        140_S64_L001_R1_001
        46.4%
        35%
        5.9
        141_S61_L001_R1_001
        35.0%
        35%
        2.0
        156_S66_L001_R1_001
        56.4%
        33%
        5.2
        159_S68_L001_R1_001
        57.8%
        33%
        9.2
        161_S57_L001_R1_001
        45.3%
        35%
        6.9
        162_S62_L001_R1_001
        46.3%
        33%
        6.3
        168_S67_L001_R1_001
        45.2%
        34%
        3.1
        169_S65_L001_R1_001
        53.4%
        34%
        11.1
        171_S58_L001_R1_001
        51.2%
        34%
        8.3
        172_S59_L001_R1_001
        41.4%
        35%
        5.0
        181_S69_L001_R1_001
        53.5%
        33%
        2.7
        183_S56_L001_R1_001
        44.6%
        35%
        9.6
        184_S55_L001_R1_001
        39.1%
        36%
        3.4
        185_S60_L001_R1_001
        43.8%
        35%
        2.2
        291_S42_L001_R1_001
        41.5%
        35%
        7.3
        292_S32_L001_R1_001
        33.4%
        39%
        5.3
        293_S11_L001_R1_001
        36.4%
        38%
        5.1
        294_S7_L001_R1_001
        43.2%
        36%
        7.4
        295_S41_L001_R1_001
        38.3%
        36%
        9.9
        296_S40_L001_R1_001
        38.3%
        35%
        12.1
        298_S25_L001_R1_001
        33.3%
        36%
        6.6
        299_S21_L001_R1_001
        34.4%
        38%
        9.8
        301_S22_L001_R1_001
        36.9%
        35%
        7.4
        302_S15_L001_R1_001
        32.8%
        36%
        10.2
        303_S17_L001_R1_001
        36.2%
        35%
        7.8
        304_S24_L001_R1_001
        33.4%
        37%
        9.0
        305_S1_L001_R1_001
        37.9%
        36%
        6.2
        306_S44_L001_R1_001
        44.0%
        34%
        8.0
        307_S45_L001_R1_001
        44.2%
        35%
        7.5
        308_S5_L001_R1_001
        47.3%
        36%
        8.2
        309_S20_L001_R1_001
        37.3%
        36%
        8.6
        311_S2_L001_R1_001
        36.2%
        36%
        7.1
        312_S28_L001_R1_001
        38.1%
        36%
        7.1
        313_S36_L001_R1_001
        41.1%
        36%
        7.0
        314_S49_L001_R1_001
        54.1%
        37%
        5.4
        315_S26_L001_R1_001
        35.9%
        36%
        9.0
        316_S9_L001_R1_001
        37.6%
        36%
        9.8
        317_S33_L001_R1_001
        32.4%
        37%
        5.9
        318_S6_L001_R1_001
        38.3%
        35%
        6.3
        319_S52_L001_R1_001
        57.0%
        35%
        8.0
        321_S29_L001_R1_001
        32.5%
        36%
        6.2
        322_S8_L001_R1_001
        39.0%
        36%
        7.6
        323_S39_L001_R1_001
        34.1%
        36%
        10.2
        324_S47_L001_R1_001
        43.3%
        35%
        8.5
        325_S13_L001_R1_001
        32.6%
        35%
        7.9
        326_S38_L001_R1_001
        41.4%
        35%
        9.1
        327_S37_L001_R1_001
        37.6%
        36%
        5.9
        328_S12_L001_R1_001
        31.3%
        38%
        12.9
        329_S46_L001_R1_001
        43.7%
        35%
        8.5
        331_S53_L001_R1_001
        56.5%
        39%
        6.9
        332_S48_L001_R1_001
        47.0%
        35%
        5.6
        333_S30_L001_R1_001
        36.5%
        35%
        5.4
        334_S50_L001_R1_001
        54.6%
        36%
        5.8
        335_S31_L001_R1_001
        35.5%
        36%
        5.4
        336_S51_L001_R1_001
        53.8%
        35%
        7.8
        337_S35_L001_R1_001
        45.0%
        35%
        10.8
        338_S4_L001_R1_001
        40.5%
        35%
        6.5
        339_S10_L001_R1_001
        43.2%
        36%
        7.4
        341_S19_L001_R1_001
        36.3%
        37%
        5.7
        342_S23_L001_R1_001
        40.9%
        36%
        8.9
        343_S14_L001_R1_001
        32.3%
        37%
        7.9
        344_S27_L001_R1_001
        47.1%
        38%
        11.1
        345_S16_L001_R1_001
        28.4%
        35%
        5.8
        346_S18_L001_R1_001
        33.4%
        37%
        5.9
        347_S43_L001_R1_001
        41.5%
        34%
        6.7
        348_S3_L001_R1_001
        42.1%
        36%
        7.3
        349_S34_L001_R1_001
        32.6%
        37%
        5.9
        34_S68_L002_R1_001
        74.4%
        37%
        6.9
        35_S72_L002_R1_001
        69.7%
        48%
        2.7
        37_S70_L002_R1_001
        83.3%
        39%
        7.6
        39_S52_L002_R1_001
        45.7%
        36%
        6.0
        401_S10_L002_R1_001
        65.3%
        38%
        4.1
        402_S5_L002_R1_001
        46.0%
        37%
        5.8
        403_S30_L002_R1_001
        38.8%
        36%
        6.8
        404_S42_L002_R1_001
        54.7%
        35%
        7.0
        411_S9_L002_R1_001
        49.1%
        35%
        6.6
        412_S74_L002_R1_001
        90.7%
        40%
        7.1
        413_S38_L002_R1_001
        49.3%
        35%
        6.4
        414_S49_L002_R1_001
        37.2%
        39%
        5.6
        41_S62_L002_R1_001
        72.6%
        37%
        8.3
        421_S22_L002_R1_001
        40.3%
        37%
        7.6
        431b_S8_L002_R1_001
        54.4%
        37%
        6.4
        432_S75_L002_R1_001
        91.1%
        43%
        6.0
        434_S55_L002_R1_001
        56.8%
        33%
        7.1
        43_S46_L002_R1_001
        44.6%
        36%
        7.3
        441_S73_L002_R1_001
        90.6%
        45%
        9.2
        442b_S60_L002_R1_001
        57.5%
        35%
        5.4
        443_S36_L002_R1_001
        53.6%
        34%
        6.2
        444_S34_L002_R1_001
        55.5%
        32%
        8.1
        445_S45_L002_R1_001
        40.3%
        36%
        6.6
        44_S71_L002_R1_001
        66.3%
        51%
        1.4
        451_S28_L002_R1_001
        47.0%
        35%
        9.1
        452b_S2_L002_R1_001
        51.2%
        37%
        5.6
        453_S12_L002_R1_001
        40.2%
        36%
        5.3
        45_S63_L002_R1_001
        67.6%
        36%
        7.2
        461b_S31_L002_R1_001
        46.6%
        34%
        4.6
        462b_S64_L002_R1_001
        57.8%
        34%
        10.2
        46_S66_L002_R1_001
        74.5%
        36%
        7.6
        471b_S51_L002_R1_001
        46.8%
        35%
        6.4
        472b_S48_L002_R1_001
        46.6%
        35%
        6.3
        473_S20_L002_R1_001
        46.6%
        35%
        6.6
        474_S14_L002_R1_001
        50.0%
        34%
        8.5
        475_S16_L002_R1_001
        48.1%
        34%
        7.4
        476_S17_L002_R1_001
        49.8%
        31%
        4.7
        477_S37_L002_R1_001
        44.1%
        37%
        6.6
        47_S58_L002_R1_001
        49.9%
        36%
        5.5
        481_S57_L002_R1_001
        49.4%
        35%
        7.7
        482_S25_L002_R1_001
        52.8%
        32%
        5.4
        483_S7_L002_R1_001
        52.6%
        36%
        6.1
        484_S43_L002_R1_001
        46.7%
        37%
        5.2
        485_S21_L002_R1_001
        42.9%
        36%
        7.1
        487_S6_L002_R1_001
        49.3%
        36%
        7.4
        488_S26_L002_R1_001
        52.2%
        31%
        6.9
        489_S35_L002_R1_001
        42.2%
        38%
        7.4
        490_S19_L002_R1_001
        40.6%
        36%
        6.0
        491_S50_L002_R1_001
        55.0%
        32%
        6.5
        492_S40_L002_R1_001
        53.9%
        33%
        8.0
        506_S47_L002_R1_001
        60.9%
        32%
        5.9
        513_S56_L002_R1_001
        41.6%
        35%
        6.4
        521_S65_L002_R1_001
        73.3%
        35%
        11.8
        522_S1_L002_R1_001
        56.0%
        34%
        5.9
        523_S4_L002_R1_001
        58.6%
        35%
        7.1
        524_S11_L002_R1_001
        49.5%
        34%
        6.2
        525_S18_L002_R1_001
        36.7%
        37%
        5.5
        526_S15_L002_R1_001
        41.2%
        36%
        5.3
        527_S39_L002_R1_001
        51.0%
        35%
        8.0
        528_S27_L002_R1_001
        45.4%
        34%
        8.8
        529_S53_L002_R1_001
        58.5%
        32%
        7.0
        531_S44_L002_R1_001
        39.9%
        37%
        5.1
        532_S33_L002_R1_001
        48.3%
        34%
        5.1
        533_S61_L002_R1_001
        50.0%
        36%
        5.4
        541_S41_L002_R1_001
        55.3%
        35%
        7.2
        542_S3_L002_R1_001
        65.0%
        34%
        6.3
        543_S29_L002_R1_001
        39.5%
        34%
        4.0
        551_S24_L002_R1_001
        47.6%
        34%
        7.6
        552b_S54_L002_R1_001
        49.5%
        35%
        4.4
        553_S23_L002_R1_001
        37.2%
        36%
        4.9
        554_S13_L002_R1_001
        41.5%
        37%
        8.1
        561_S69_L002_R1_001
        85.9%
        34%
        6.4
        562_S77_L002_R1_001
        74.7%
        39%
        16.1
        563_S59_L002_R1_001
        65.4%
        34%
        8.3
        564_S32_L002_R1_001
        40.2%
        36%
        4.6
        565_S67_L002_R1_001
        74.7%
        40%
        5.6
        571_S76_L002_R1_001
        79.1%
        36%
        1.4

        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        Sequence Length Distribution

        All samples have sequences of a single length (100bp).

        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        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.

        Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).


        Status Checks

        Status for each FastQC section showing whether results seem entirely normal (green), slightly abnormal (orange) or very unusual (red).

        FastQC assigns a status for each section of the report. These give a quick evaluation of whether the results of the analysis seem entirely normal (green), slightly abnormal (orange) or very unusual (red).

        It is important to stress that although the analysis results appear to give a pass/fail result, these evaluations must be taken in the context of what you expect from your library. A 'normal' sample as far as FastQC is concerned is random and diverse. Some experiments may be expected to produce libraries which are biased in particular ways. You should treat the summary evaluations therefore as pointers to where you should concentrate your attention and understand why your library may not look random and diverse.

        Specific guidance on how to interpret the output of each module can be found in the relevant report section, or in the FastQC help.

        In this heatmap, we summarise all of these into a single heatmap for a quick overview. Note that not all FastQC sections have plots in MultiQC reports, but all status checks are shown in this heatmap.

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