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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 1.9

        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 2022-03-16, 22:46 based on data in: /scratch/gencore/logs/html/H75JHBGXL/merged


        General Statistics

        Showing 72/72 rows and 3/5 columns.
        Sample Name% Dups% GCM Seqs
        H75JHBGXL_n01_new3064Mar1
        60.3%
        44%
        4.5
        H75JHBGXL_n01_new3064Mar2
        25.5%
        42%
        0.3
        H75JHBGXL_n01_new3064Mar3
        67.3%
        43%
        5.9
        H75JHBGXL_n01_new4692Mar1
        30.3%
        43%
        7.1
        H75JHBGXL_n01_new4692Mar2
        37.1%
        43%
        6.8
        H75JHBGXL_n01_new4692Mar3
        36.1%
        42%
        8.0
        H75JHBGXL_n01_new4753Mar1
        31.1%
        42%
        6.2
        H75JHBGXL_n01_new4753Mar2
        45.7%
        43%
        7.0
        H75JHBGXL_n01_new4753Mar3
        39.7%
        44%
        5.4
        H75JHBGXL_n01_new5890Mar1
        33.5%
        42%
        7.5
        H75JHBGXL_n01_new5890Mar2
        26.6%
        42%
        2.7
        H75JHBGXL_n01_new5890Mar3
        20.6%
        42%
        1.6
        H75JHBGXL_n01_new5900Mar1
        36.4%
        44%
        4.3
        H75JHBGXL_n01_new5900Mar2
        40.1%
        45%
        0.9
        H75JHBGXL_n01_new6004Mar1
        35.9%
        42%
        4.5
        H75JHBGXL_n01_new6004Mar3
        35.9%
        43%
        4.4
        H75JHBGXL_n01_new6405Mar1
        34.3%
        43%
        4.0
        H75JHBGXL_n01_new6405Mar2
        33.4%
        42%
        5.5
        H75JHBGXL_n01_new6405Mar3
        38.7%
        43%
        3.8
        H75JHBGXL_n01_newEVMar1
        14.7%
        43%
        0.8
        H75JHBGXL_n01_newEVMar2
        27.8%
        44%
        1.8
        H75JHBGXL_n01_newEVMar3
        35.7%
        44%
        6.1
        H75JHBGXL_n01_newEVMar4
        73.5%
        45%
        5.8
        H75JHBGXL_n01_newRS1
        15.4%
        38%
        7.4
        H75JHBGXL_n01_newRS10
        15.3%
        37%
        9.2
        H75JHBGXL_n01_newRS11
        15.8%
        38%
        8.5
        H75JHBGXL_n01_newRS12
        20.0%
        39%
        8.1
        H75JHBGXL_n01_newRS13
        18.0%
        38%
        8.4
        H75JHBGXL_n01_newRS14
        15.7%
        37%
        8.0
        H75JHBGXL_n01_newRS15
        15.7%
        37%
        8.8
        H75JHBGXL_n01_newRS16
        17.0%
        39%
        8.0
        H75JHBGXL_n01_newRS17
        15.5%
        37%
        8.9
        H75JHBGXL_n01_newRS18
        17.4%
        38%
        9.1
        H75JHBGXL_n01_newRS19
        15.3%
        37%
        7.8
        H75JHBGXL_n01_newRS2
        16.3%
        39%
        8.5
        H75JHBGXL_n01_newRS20
        16.8%
        37%
        9.6
        H75JHBGXL_n01_newRS21
        16.1%
        37%
        8.4
        H75JHBGXL_n01_newRS22
        17.6%
        37%
        9.1
        H75JHBGXL_n01_newRS23
        14.7%
        37%
        7.1
        H75JHBGXL_n01_newRS24
        14.6%
        38%
        6.3
        H75JHBGXL_n01_newRS25
        15.4%
        38%
        7.7
        H75JHBGXL_n01_newRS26
        16.3%
        38%
        8.1
        H75JHBGXL_n01_newRS27
        17.1%
        40%
        7.3
        H75JHBGXL_n01_newRS28
        16.5%
        38%
        7.5
        H75JHBGXL_n01_newRS29
        14.7%
        38%
        7.2
        H75JHBGXL_n01_newRS3
        16.0%
        38%
        7.7
        H75JHBGXL_n01_newRS30
        16.8%
        39%
        8.7
        H75JHBGXL_n01_newRS31
        16.1%
        37%
        7.9
        H75JHBGXL_n01_newRS32
        12.9%
        41%
        2.7
        H75JHBGXL_n01_newRS33
        15.3%
        37%
        8.8
        H75JHBGXL_n01_newRS34
        14.3%
        37%
        8.7
        H75JHBGXL_n01_newRS35
        14.7%
        38%
        7.1
        H75JHBGXL_n01_newRS36
        16.6%
        38%
        7.9
        H75JHBGXL_n01_newRS37
        16.5%
        39%
        7.8
        H75JHBGXL_n01_newRS38
        17.6%
        37%
        8.7
        H75JHBGXL_n01_newRS39
        20.6%
        39%
        8.5
        H75JHBGXL_n01_newRS4
        15.9%
        38%
        7.8
        H75JHBGXL_n01_newRS40
        16.9%
        38%
        9.9
        H75JHBGXL_n01_newRS41
        16.9%
        37%
        8.2
        H75JHBGXL_n01_newRS42
        15.9%
        37%
        8.3
        H75JHBGXL_n01_newRS43
        16.8%
        38%
        8.9
        H75JHBGXL_n01_newRS44
        17.4%
        38%
        8.1
        H75JHBGXL_n01_newRS45
        17.3%
        39%
        7.8
        H75JHBGXL_n01_newRS46
        19.3%
        37%
        14.2
        H75JHBGXL_n01_newRS47
        18.6%
        37%
        16.1
        H75JHBGXL_n01_newRS48
        16.5%
        38%
        8.2
        H75JHBGXL_n01_newRS5
        14.7%
        38%
        8.5
        H75JHBGXL_n01_newRS6
        16.3%
        39%
        8.6
        H75JHBGXL_n01_newRS7
        16.5%
        39%
        7.9
        H75JHBGXL_n01_newRS8
        16.6%
        40%
        8.0
        H75JHBGXL_n01_newRS9
        15.0%
        37%
        7.5
        H75JHBGXL_n01_undetermined
        83.9%
        43%
        69.3

        Barcodes of Undetermined Reads


        We have determined the barcodes of your undetermined reads (reads containing a barcode that you did not encode in your metadata). Here are the top 20 barcodes belonging to the undetermined reads. The full list is available here.

        Showing 20/20 rows and 2/2 columns.
        Barcode Sequence(s)CountFrequency (%)
        GGGGGG
        63311066.0
        91.3
        GGGGGC
        105549.0
        0.1
        GCGGGG
        93172.0
        0.1
        GGGGCG
        79152.0
        0.1
        GGGGTG
        69325.0
        0.1
        GGGCGG
        68916.0
        0.1
        GGCGGG
        65883.0
        0.1
        CGGGGG
        57628.0
        0.1
        GTGGGG
        50889.0
        0.1
        TTATGA
        48259.0
        0.1
        TGGGGG
        38044.0
        0.1
        NNNNNN
        35670.0
        0.1
        GGGTGG
        33775.0
        0.1
        TGGTAA
        31239.0
        0.1
        ATTGTA
        29191.0
        0.0
        CTGCTA
        27809.0
        0.0
        TTGTAA
        27375.0
        0.0
        AAAAAA
        26886.0
        0.0
        GGGGCC
        26871.0
        0.0
        CTCTCG
        26679.0
        0.0

        Demultiplexing Report


        Total Read Count: Total number of PF (Passing Filter) reads in this library.
        Portion: The proportion of reads that represent the individual library in the entire Library Pool.

        Showing 72/72 rows and 2/2 columns.
        LibraryTotal Read CountPortion (%)
        undetermined_library
        69325569
        12.0
        newEVMar1
        808172
        0.1
        newEVMar2
        1790838
        0.3
        newEVMar3
        6055444
        1.1
        new3064Mar1
        4511760
        0.8
        new3064Mar2
        328702
        0.1
        new3064Mar3
        5918944
        1.0
        new4692Mar1
        7106585
        1.2
        new4692Mar2
        6811971
        1.2
        new4692Mar3
        8042425
        1.4
        new4753Mar1
        6201169
        1.1
        new4753Mar2
        7030294
        1.2
        new4753Mar3
        5390110
        0.9
        new6405Mar1
        4033873
        0.7
        new6405Mar2
        5543881
        1.0
        new6405Mar3
        3827617
        0.7
        new5890Mar1
        7491092
        1.3
        new5890Mar2
        2673706
        0.5
        new5890Mar3
        1561926
        0.3
        new6004Mar1
        4486981
        0.8
        new6004Mar3
        4403889
        0.8
        new5900Mar1
        4258757
        0.7
        new5900Mar2
        929697
        0.2
        newEVMar4
        5842365
        1.0
        newRS1
        7441224
        1.3
        newRS2
        8501064
        1.5
        newRS3
        7686292
        1.3
        newRS4
        7808085
        1.4
        newRS5
        8543504
        1.5
        newRS6
        8619028
        1.5
        newRS7
        7910348
        1.4
        newRS8
        7999526
        1.4
        newRS9
        7495008
        1.3
        newRS10
        9227561
        1.6
        newRS11
        8457172
        1.5
        newRS12
        8146580
        1.4
        newRS13
        8416744
        1.5
        newRS14
        8005079
        1.4
        newRS15
        8792714
        1.5
        newRS16
        8032302
        1.4
        newRS17
        8937094
        1.6
        newRS18
        9083125
        1.6
        newRS19
        7849993
        1.4
        newRS20
        9564525
        1.7
        newRS21
        8391068
        1.5
        newRS22
        9132802
        1.6
        newRS23
        7094454
        1.2
        newRS24
        6346985
        1.1
        newRS25
        7665499
        1.3
        newRS26
        8059479
        1.4
        newRS27
        7340001
        1.3
        newRS28
        7480221
        1.3
        newRS29
        7215893
        1.3
        newRS30
        8737765
        1.5
        newRS31
        7855462
        1.4
        newRS32
        2690092
        0.5
        newRS33
        8759398
        1.5
        newRS34
        8732878
        1.5
        newRS35
        7108756
        1.2
        newRS36
        7881062
        1.4
        newRS37
        7831631
        1.4
        newRS38
        8693808
        1.5
        newRS39
        8484699
        1.5
        newRS40
        9855222
        1.7
        newRS41
        8233896
        1.4
        newRS42
        8318734
        1.4
        newRS43
        8910709
        1.5
        newRS44
        8088714
        1.4
        newRS45
        7790109
        1.4
        newRS46
        14228549
        2.5
        newRS47
        16064211
        2.8
        newRS48
        8174338
        1.4

        Run Statistics

        Showing 1/1 rows and 4/4 columns.
        Number of LanesTotal # of Single-End ReadsTotal # PF Reads% Undetermined% PhiX Aligned
        4.0
        686041440
        576059170
        12.0
        10.3

        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.

        loading..

        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.

        loading..

        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.

        loading..

        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.

        loading..

        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.

        loading..

        Sequence Length Distribution

        All samples have sequences of a single length (76bp).

        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.

        loading..

        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.

        72 samples had less than 1% of reads made up of overrepresented sequences

        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.

        loading..

        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.

        loading..