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        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 1.11

        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 2021-12-10, 13:01 based on data in: /nfs/users/bi/projects/external/Anna_Ferrer/sequencing_analysis/Anna_Ferrer/2021-11-29-ampli_seq/analysis/report


        General Statistics

        Showing 165/165 rows and 13/18 columns.
        Sample NameM Reads Mapped% Dups% GCM Seqs% Trimmed% Dups% GCLengthM Seqs% Dups% GCLengthM Seqs
        06326AAC-E02X5A_S1_L001
        89.0%
        55%
        274 bp
        0.6
        06326AAC-E02X5A_S1_L001.assembled
        91.7%
        55%
        421 bp
        0.6
        06326AAC-E02X5A_S1_L001_R1_001
        89.1%
        54%
        0.6
        17.4%
        06326AAC-E02X5A_S1_L001_R2_001
        85.7%
        54%
        0.6
        06326AAC-E02X5A_S1_L001_s.stat
        0.5
        06327AAC-E03X5A_S2_L001
        89.7%
        56%
        289 bp
        0.7
        06327AAC-E03X5A_S2_L001.assembled
        93.9%
        56%
        439 bp
        0.7
        06327AAC-E03X5A_S2_L001_R1_001
        92.6%
        56%
        0.7
        7.6%
        06327AAC-E03X5A_S2_L001_R2_001
        89.7%
        56%
        0.7
        06327AAC-E03X5A_S2_L001_s.stat
        0.7
        06328AAC-E04X5A_S3_L001
        88.0%
        56%
        285 bp
        0.5
        06328AAC-E04X5A_S3_L001.assembled
        93.4%
        56%
        438 bp
        0.5
        06328AAC-E04X5A_S3_L001_R1_001
        91.6%
        56%
        0.5
        9.3%
        06328AAC-E04X5A_S3_L001_R2_001
        88.0%
        55%
        0.5
        06328AAC-E04X5A_S3_L001_s.stat
        0.5
        06329AAC-E05X5A_S4_L001
        92.4%
        56%
        287 bp
        0.6
        06329AAC-E05X5A_S4_L001.assembled
        93.9%
        56%
        441 bp
        0.6
        06329AAC-E05X5A_S4_L001_R1_001
        92.3%
        55%
        0.6
        8.6%
        06329AAC-E05X5A_S4_L001_R2_001
        89.2%
        55%
        0.6
        06329AAC-E05X5A_S4_L001_s.stat
        0.5
        06330AAC-E06X5A_S5_L001
        87.7%
        56%
        277 bp
        0.8
        06330AAC-E06X5A_S5_L001.assembled
        92.8%
        56%
        426 bp
        0.7
        06330AAC-E06X5A_S5_L001_R1_001
        90.6%
        55%
        0.8
        15.0%
        06330AAC-E06X5A_S5_L001_R2_001
        87.8%
        55%
        0.8
        06330AAC-E06X5A_S5_L001_s.stat
        0.6
        06331AAC-E07X5A_S6_L001
        89.1%
        55%
        286 bp
        0.7
        06331AAC-E07X5A_S6_L001.assembled
        93.1%
        56%
        436 bp
        0.6
        06331AAC-E07X5A_S6_L001_R1_001
        91.6%
        55%
        0.7
        10.0%
        06331AAC-E07X5A_S6_L001_R2_001
        89.1%
        55%
        0.7
        06331AAC-E07X5A_S6_L001_s.stat
        0.5
        06332AAC-E08X5A_S7_L001
        91.3%
        56%
        279 bp
        0.7
        06332AAC-E08X5A_S7_L001.assembled
        93.6%
        55%
        435 bp
        0.7
        06332AAC-E08X5A_S7_L001_R1_001
        91.2%
        55%
        0.7
        12.9%
        06332AAC-E08X5A_S7_L001_R2_001
        88.3%
        55%
        0.7
        06332AAC-E08X5A_S7_L001_s.stat
        0.6
        06333AAC-E17X5A_S8_L001
        91.1%
        56%
        280 bp
        0.5
        06333AAC-E17X5A_S8_L001.assembled
        93.6%
        55%
        445 bp
        0.5
        06333AAC-E17X5A_S8_L001_R1_001
        91.0%
        55%
        0.5
        11.4%
        06333AAC-E17X5A_S8_L001_R2_001
        87.6%
        54%
        0.5
        06333AAC-E17X5A_S8_L001_s.stat
        0.4
        06334AAC-E18X5A_S9_L001
        92.1%
        56%
        285 bp
        0.6
        06334AAC-E18X5A_S9_L001.assembled
        93.7%
        56%
        438 bp
        0.6
        06334AAC-E18X5A_S9_L001_R1_001
        91.9%
        56%
        0.6
        9.5%
        06334AAC-E18X5A_S9_L001_R2_001
        88.4%
        55%
        0.6
        06334AAC-E18X5A_S9_L001_s.stat
        0.5
        06335AAC-E19X5A_S10_L001
        93.1%
        56%
        287 bp
        0.8
        06335AAC-E19X5A_S10_L001.assembled
        94.5%
        56%
        441 bp
        0.7
        06335AAC-E19X5A_S10_L001_R1_001
        92.9%
        55%
        0.8
        8.4%
        06335AAC-E19X5A_S10_L001_R2_001
        89.6%
        55%
        0.8
        06335AAC-E19X5A_S10_L001_s.stat
        0.7
        06336AAC-E21X5A_S11_L001
        87.7%
        56%
        285 bp
        0.6
        06336AAC-E21X5A_S11_L001.assembled
        92.9%
        56%
        431 bp
        0.6
        06336AAC-E21X5A_S11_L001_R1_001
        91.1%
        56%
        0.6
        11.0%
        06336AAC-E21X5A_S11_L001_R2_001
        87.7%
        55%
        0.6
        06336AAC-E21X5A_S11_L001_s.stat
        0.5
        06337AAC-E22X5A_S12_L001
        88.9%
        55%
        285 bp
        0.7
        06337AAC-E22X5A_S12_L001.assembled
        94.0%
        56%
        435 bp
        0.6
        06337AAC-E22X5A_S12_L001_R1_001
        92.3%
        55%
        0.7
        10.7%
        06337AAC-E22X5A_S12_L001_R2_001
        88.9%
        55%
        0.7
        06337AAC-E22X5A_S12_L001_s.stat
        0.6
        06338AAC-E23X5A_S13_L001
        88.3%
        55%
        287 bp
        0.6
        06338AAC-E23X5A_S13_L001.assembled
        93.1%
        55%
        431 bp
        0.6
        06338AAC-E23X5A_S13_L001_R1_001
        91.5%
        54%
        0.6
        10.6%
        06338AAC-E23X5A_S13_L001_R2_001
        88.3%
        54%
        0.6
        06338AAC-E23X5A_S13_L001_s.stat
        0.5
        06339AAC-E24X5A_S14_L001
        87.8%
        55%
        285 bp
        0.6
        06339AAC-E24X5A_S14_L001.assembled
        93.2%
        56%
        436 bp
        0.5
        06339AAC-E24X5A_S14_L001_R1_001
        91.4%
        55%
        0.6
        10.1%
        06339AAC-E24X5A_S14_L001_R2_001
        87.8%
        55%
        0.6
        06339AAC-E24X5A_S14_L001_s.stat
        0.5
        06340AAC-E02X6A_S15_L001
        87.6%
        56%
        277 bp
        0.6
        06340AAC-E02X6A_S15_L001.assembled
        93.9%
        56%
        436 bp
        0.6
        06340AAC-E02X6A_S15_L001_R1_001
        91.2%
        55%
        0.6
        12.4%
        06340AAC-E02X6A_S15_L001_R2_001
        87.8%
        55%
        0.6
        06340AAC-E02X6A_S15_L001_s.stat
        0.6
        06341AAC-E04X6A_S16_L001
        90.0%
        55%
        279 bp
        0.5
        06341AAC-E04X6A_S16_L001.assembled
        92.4%
        55%
        435 bp
        0.5
        06341AAC-E04X6A_S16_L001_R1_001
        89.9%
        55%
        0.5
        13.2%
        06341AAC-E04X6A_S16_L001_R2_001
        86.7%
        55%
        0.5
        06341AAC-E04X6A_S16_L001_s.stat
        0.4
        06342AAC-E08X6A_S17_L001
        86.7%
        55%
        278 bp
        0.5
        06342AAC-E08X6A_S17_L001.assembled
        92.7%
        55%
        437 bp
        0.4
        06342AAC-E08X6A_S17_L001_R1_001
        90.0%
        55%
        0.5
        12.2%
        06342AAC-E08X6A_S17_L001_R2_001
        86.7%
        55%
        0.5
        06342AAC-E08X6A_S17_L001_s.stat
        0.4
        06343AAC-E12X6A_S18_L001
        87.1%
        55%
        280 bp
        0.5
        06343AAC-E12X6A_S18_L001.assembled
        93.2%
        55%
        438 bp
        0.4
        06343AAC-E12X6A_S18_L001_R1_001
        90.5%
        55%
        0.5
        10.7%
        06343AAC-E12X6A_S18_L001_R2_001
        87.1%
        55%
        0.5
        06343AAC-E12X6A_S18_L001_s.stat
        0.4
        06344AAC-E14X6A_S19_L001
        91.0%
        55%
        279 bp
        0.6
        06344AAC-E14X6A_S19_L001.assembled
        93.2%
        55%
        431 bp
        0.6
        06344AAC-E14X6A_S19_L001_R1_001
        90.8%
        54%
        0.6
        13.2%
        06344AAC-E14X6A_S19_L001_R2_001
        87.7%
        54%
        0.6
        06344AAC-E14X6A_S19_L001_s.stat
        0.5
        06345AAC-E15X6A_S20_L001
        87.3%
        55%
        284 bp
        0.5
        06345AAC-E15X6A_S20_L001.assembled
        93.0%
        55%
        436 bp
        0.5
        06345AAC-E15X6A_S20_L001_R1_001
        90.8%
        55%
        0.5
        10.1%
        06345AAC-E15X6A_S20_L001_R2_001
        87.3%
        55%
        0.5
        06345AAC-E15X6A_S20_L001_s.stat
        0.4
        06346AAC-E16X6A_S21_L001
        88.3%
        55%
        269 bp
        0.8
        06346AAC-E16X6A_S21_L001.assembled
        90.8%
        55%
        406 bp
        0.7
        06346AAC-E16X6A_S21_L001_R1_001
        88.3%
        54%
        0.8
        21.7%
        06346AAC-E16X6A_S21_L001_R2_001
        84.6%
        54%
        0.8
        06346AAC-E16X6A_S21_L001_s.stat
        0.5
        06347AAC-E17X6A_S22_L001
        91.3%
        55%
        281 bp
        0.8
        06347AAC-E17X6A_S22_L001.assembled
        93.6%
        55%
        436 bp
        0.7
        06347AAC-E17X6A_S22_L001_R1_001
        91.2%
        55%
        0.8
        11.4%
        06347AAC-E17X6A_S22_L001_R2_001
        87.9%
        55%
        0.8
        06347AAC-E17X6A_S22_L001_s.stat
        0.7
        06348AAC-E18X6A_S23_L001
        91.0%
        56%
        281 bp
        0.8
        06348AAC-E18X6A_S23_L001.assembled
        93.0%
        56%
        431 bp
        0.7
        06348AAC-E18X6A_S23_L001_R1_001
        90.8%
        55%
        0.8
        12.3%
        06348AAC-E18X6A_S23_L001_R2_001
        87.7%
        55%
        0.8
        06348AAC-E18X6A_S23_L001_s.stat
        0.6
        06349AAC-E19X6A_S24_L001
        87.1%
        56%
        281 bp
        0.6
        06349AAC-E19X6A_S24_L001.assembled
        92.3%
        56%
        427 bp
        0.6
        06349AAC-E19X6A_S24_L001_R1_001
        90.3%
        55%
        0.6
        13.4%
        06349AAC-E19X6A_S24_L001_R2_001
        87.2%
        55%
        0.6
        06349AAC-E19X6A_S24_L001_s.stat
        0.5
        06350AAC-E20X6A_S25_L001
        87.4%
        55%
        282 bp
        0.6
        06350AAC-E20X6A_S25_L001.assembled
        92.4%
        56%
        428 bp
        0.6
        06350AAC-E20X6A_S25_L001_R1_001
        90.4%
        55%
        0.6
        12.4%
        06350AAC-E20X6A_S25_L001_R2_001
        87.4%
        55%
        0.6
        06350AAC-E20X6A_S25_L001_s.stat
        0.5
        06351AAC-E21X6A_S26_L001
        91.9%
        56%
        286 bp
        0.8
        06351AAC-E21X6A_S26_L001.assembled
        93.5%
        55%
        434 bp
        0.8
        06351AAC-E21X6A_S26_L001_R1_001
        91.8%
        55%
        0.8
        10.2%
        06351AAC-E21X6A_S26_L001_R2_001
        88.8%
        55%
        0.8
        06351AAC-E21X6A_S26_L001_s.stat
        0.7
        06352AAC-E23X6A_S27_L001
        90.8%
        56%
        277 bp
        0.6
        06352AAC-E23X6A_S27_L001.assembled
        93.0%
        56%
        428 bp
        0.6
        06352AAC-E23X6A_S27_L001_R1_001
        90.7%
        55%
        0.6
        14.3%
        06352AAC-E23X6A_S27_L001_R2_001
        87.7%
        55%
        0.6
        06352AAC-E23X6A_S27_L001_s.stat
        0.5
        06353AAC-E12X5A_S28_L001
        85.6%
        54%
        267 bp
        1.0
        06353AAC-E12X5A_S28_L001.assembled
        90.9%
        55%
        396 bp
        0.9
        06353AAC-E12X5A_S28_L001_R1_001
        88.5%
        53%
        1.0
        23.8%
        06353AAC-E12X5A_S28_L001_R2_001
        86.0%
        54%
        1.0
        06353AAC-E12X5A_S28_L001_s.stat
        0.7
        06354AAC-E13X5A_S29_L001
        84.7%
        55%
        266 bp
        0.8
        06354AAC-E13X5A_S29_L001.assembled
        91.2%
        55%
        401 bp
        0.7
        06354AAC-E13X5A_S29_L001_R1_001
        88.4%
        54%
        0.8
        23.8%
        06354AAC-E13X5A_S29_L001_R2_001
        85.0%
        54%
        0.8
        06354AAC-E13X5A_S29_L001_s.stat
        0.5
        06355AAC-E15X5A_S30_L001
        86.1%
        55%
        268 bp
        0.7
        06355AAC-E15X5A_S30_L001.assembled
        88.9%
        55%
        394 bp
        0.6
        06355AAC-E15X5A_S30_L001_R1_001
        86.1%
        54%
        0.7
        23.7%
        06355AAC-E15X5A_S30_L001_R2_001
        83.0%
        54%
        0.7
        06355AAC-E15X5A_S30_L001_s.stat
        0.5
        06356AAC-E26X6A_S31_L001
        91.6%
        56%
        286 bp
        0.7
        06356AAC-E26X6A_S31_L001.assembled
        93.3%
        56%
        436 bp
        0.7
        06356AAC-E26X6A_S31_L001_R1_001
        91.6%
        56%
        0.7
        9.7%
        06356AAC-E26X6A_S31_L001_R2_001
        88.7%
        55%
        0.7
        06356AAC-E26X6A_S31_L001_s.stat
        0.6
        06357AAC-E30X6A_S32_L001
        91.5%
        56%
        285 bp
        0.7
        06357AAC-E30X6A_S32_L001.assembled
        93.3%
        56%
        437 bp
        0.7
        06357AAC-E30X6A_S32_L001_R1_001
        91.5%
        55%
        0.7
        10.2%
        06357AAC-E30X6A_S32_L001_R2_001
        88.3%
        55%
        0.7
        06357AAC-E30X6A_S32_L001_s.stat
        0.6
        06358AAC-E31X6A_S33_L001
        85.3%
        55%
        244 bp
        0.8
        06358AAC-E31X6A_S33_L001.assembled
        93.3%
        55%
        435 bp
        0.6
        06358AAC-E31X6A_S33_L001_R1_001
        88.9%
        54%
        0.8
        25.4%
        06358AAC-E31X6A_S33_L001_R2_001
        85.6%
        54%
        0.8
        06358AAC-E31X6A_S33_L001_s.stat
        0.5

        FastQC (raw)

        FastQC (raw) This section of the report shows FastQC results of raw data

        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 (301bp).

        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.

        loading..

        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..

        Skewer

        Skewer is an adapter trimming tool specially designed for processing next-generation sequencing (NGS) paired-end sequences.

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        FastQC (trimmed)

        FastQC (trimmed) This section of the report shows FastQC results after adapter trimming.

        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

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

        loading..

        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.

        loading..

        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%

        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..

        FastQC (merged)

        FastQC (merged) This section of the report shows FastQC results after merging of trimmed reads

        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

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

        loading..

        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.

        loading..

        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%

        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..

        Samtools

        Samtools is a suite of programs for interacting with high-throughput sequencing data.

        Samtools Flagstat

        This module parses the output from samtools flagstat. All numbers in millions.

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