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

        Note that additional data was saved in multiqc_GRCz11_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

        These samples were run by seq2science v0.6.1, a tool for easy preprocessing of NGS data.

        Take a look at our docs for info about how to use this report to the fullest.

        Workflow
        rna-seq
        Date
        January 13, 2022
        Project
        pt7
        Contact E-mail
        tessa.dewijs2@ru.nl

        Report generated on 2022-01-14, 09:33 based on data in:

        Change sample names:


        General Statistics

        Showing 55/55 rows and 11/21 columns.
        Sample Name% DuplicationGC content% PF% AdapterInsert Size% Dups% MappedM Total seqsGenome coverageM Genome readsM MT genome reads
        GSM4079004
        42.4%
        49.3%
        100.0%
        60.5%
        100.0%
        34.1
        1.3 X
        35.2
        0.3
        GSM4079005
        41.4%
        49.3%
        100.0%
        60.1%
        100.0%
        34.5
        1.3 X
        35.9
        0.2
        GSM4079006
        42.6%
        49.4%
        100.0%
        61.0%
        100.0%
        36.7
        1.4 X
        39.1
        0.2
        GSM4079007
        44.2%
        49.2%
        100.0%
        63.1%
        100.0%
        38.7
        1.5 X
        42.4
        0.4
        GSM4079008
        44.4%
        48.7%
        100.0%
        64.6%
        100.0%
        37.1
        1.5 X
        41.4
        0.4
        GSM4079009
        45.5%
        48.9%
        100.0%
        65.5%
        100.0%
        34.2
        1.4 X
        38.2
        0.4
        GSM4079010
        44.3%
        49.2%
        100.0%
        66.6%
        100.0%
        34.5
        1.4 X
        38.5
        0.4
        GSM4079011
        39.5%
        48.2%
        99.9%
        61.7%
        100.0%
        32.0
        1.2 X
        33.0
        0.3
        GSM4079012
        43.4%
        48.2%
        100.0%
        60.8%
        100.0%
        35.8
        1.3 X
        37.0
        0.2
        GSM4079013
        43.4%
        48.4%
        100.0%
        61.6%
        100.0%
        36.7
        1.4 X
        38.8
        0.3
        GSM4079014
        44.0%
        47.9%
        100.0%
        61.8%
        100.0%
        34.9
        1.4 X
        38.3
        0.3
        GSM4079015
        44.7%
        47.7%
        100.0%
        63.3%
        100.0%
        35.1
        1.4 X
        39.3
        0.5
        GSM4079016
        45.4%
        47.6%
        100.0%
        65.4%
        100.0%
        35.1
        1.4 X
        39.4
        0.4
        GSM4079017
        45.8%
        47.2%
        99.9%
        67.0%
        100.0%
        35.8
        1.5 X
        40.1
        0.6
        GSM4079018
        46.3%
        47.6%
        99.9%
        68.2%
        100.0%
        35.6
        1.4 X
        39.7
        0.3
        GSM4079019
        39.5%
        48.0%
        99.9%
        62.7%
        100.0%
        33.6
        1.3 X
        34.5
        0.3
        GSM4079020
        39.6%
        48.3%
        99.9%
        62.1%
        100.0%
        37.1
        1.4 X
        38.3
        0.3
        GSM4079021
        44.8%
        48.5%
        99.8%
        61.9%
        100.0%
        38.4
        1.5 X
        40.3
        0.2
        GSM4079022
        42.7%
        48.0%
        100.0%
        62.7%
        100.0%
        39.6
        1.6 X
        43.2
        0.3
        GSM4079023
        34.8%
        47.4%
        100.0%
        62.5%
        100.0%
        35.4
        1.5 X
        40.0
        0.3
        GSM4079024
        39.0%
        47.8%
        99.8%
        66.9%
        100.0%
        40.7
        1.7 X
        46.1
        0.4
        GSM4079025
        35.3%
        47.3%
        100.0%
        65.9%
        100.0%
        34.0
        1.4 X
        38.5
        0.3
        GSM4079026
        36.9%
        47.7%
        100.0%
        67.9%
        100.0%
        34.8
        1.4 X
        39.1
        0.3
        GSM4079027
        42.4%
        48.2%
        100.0%
        62.8%
        100.0%
        39.0
        1.5 X
        40.0
        0.3
        GSM4079028
        40.7%
        48.1%
        100.0%
        60.2%
        100.0%
        34.7
        1.3 X
        35.9
        0.3
        GSM4079029
        45.9%
        48.0%
        100.0%
        63.4%
        100.0%
        40.8
        1.6 X
        43.6
        0.5
        GSM4079030
        45.4%
        47.8%
        100.0%
        63.1%
        100.0%
        36.7
        1.5 X
        40.4
        0.5
        GSM4079031
        46.0%
        47.6%
        100.0%
        64.3%
        100.0%
        34.9
        1.4 X
        39.2
        0.4
        GSM4079032
        46.1%
        47.8%
        100.0%
        65.9%
        100.0%
        36.3
        1.5 X
        40.7
        0.4
        GSM4079033
        48.5%
        47.6%
        99.8%
        67.2%
        100.0%
        37.0
        1.5 X
        41.6
        0.4
        GSM4079034
        49.1%
        47.8%
        99.7%
        67.6%
        100.0%
        37.3
        1.5 X
        41.8
        0.4
        GSM4282070
        41.9%
        49.2%
        100.0%
        60.5%
        100.0%
        34.8
        1.3 X
        35.8
        0.3
        GSM4282071
        41.9%
        49.2%
        99.9%
        60.1%
        100.0%
        34.7
        1.3 X
        36.1
        0.2
        GSM4282072
        41.4%
        49.5%
        100.0%
        60.5%
        100.0%
        34.7
        1.3 X
        37.1
        0.2
        GSM4282073
        43.1%
        49.2%
        100.0%
        62.4%
        100.0%
        36.4
        1.5 X
        40.0
        0.3
        GSM4282074
        45.7%
        48.8%
        100.0%
        65.3%
        100.0%
        38.0
        1.5 X
        42.4
        0.4
        GSM4282075
        44.2%
        49.1%
        100.0%
        65.0%
        100.0%
        33.9
        1.4 X
        38.0
        0.4
        GSM4282076
        41.7%
        49.1%
        99.9%
        66.7%
        100.0%
        34.9
        1.4 X
        39.1
        0.4
        GSM831503
        2.6%
        46.7%
        80.2%
        0.2%
        287 bp
        18.1%
        100.0%
        66.0
        3.6 X
        65.1
        2.9
        GSM831504
        4.9%
        46.8%
        83.3%
        0.1%
        283 bp
        22.6%
        100.0%
        98.8
        5.4 X
        97.5
        4.4
        GSM831505
        3.3%
        46.9%
        77.3%
        0.2%
        266 bp
        15.5%
        100.0%
        98.2
        5.5 X
        99.0
        2.2
        GSM831506
        4.3%
        47.0%
        80.6%
        0.1%
        260 bp
        17.1%
        100.0%
        108.3
        6.0 X
        108.9
        2.4
        GSM831507
        3.2%
        45.9%
        76.8%
        0.2%
        256 bp
        17.2%
        100.0%
        93.4
        5.5 X
        100.1
        2.6
        GSM831508
        4.5%
        46.1%
        81.1%
        0.2%
        253 bp
        19.1%
        100.0%
        106.0
        6.3 X
        113.4
        2.9
        GSM831509
        1.1%
        46.0%
        81.6%
        0.2%
        312 bp
        12.8%
        100.0%
        27.8
        1.7 X
        29.9
        0.5
        GSM831510
        1.3%
        45.9%
        83.1%
        0.2%
        308 bp
        14.1%
        100.0%
        33.9
        2.0 X
        36.5
        0.6
        GSM831511
        4.2%
        46.5%
        93.0%
        0.2%
        326 bp
        22.5%
        100.0%
        80.5
        4.8 X
        86.4
        1.4
        GSM831512
        3.3%
        46.7%
        79.6%
        0.3%
        258 bp
        26.0%
        100.0%
        81.4
        4.9 X
        88.3
        2.1
        GSM831513
        5.0%
        46.7%
        82.8%
        0.2%
        256 bp
        29.1%
        100.0%
        103.0
        6.2 X
        111.7
        2.7
        GSM831514
        3.2%
        46.8%
        79.6%
        0.2%
        259 bp
        28.4%
        100.0%
        82.8
        4.8 X
        87.6
        2.5
        GSM831515
        4.8%
        46.9%
        83.2%
        0.1%
        257 bp
        31.0%
        100.0%
        100.3
        5.9 X
        106.5
        3.1
        GSM831516
        3.3%
        46.7%
        76.3%
        0.2%
        251 bp
        30.8%
        100.0%
        86.5
        5.1 X
        91.5
        2.8
        GSM831517
        4.4%
        46.9%
        82.2%
        0.1%
        250 bp
        33.2%
        100.0%
        105.5
        6.2 X
        111.5
        3.5
        GSM831518
        5.0%
        45.7%
        69.4%
        0.1%
        423 bp
        27.4%
        100.0%
        54.9
        3.1 X
        56.4
        2.4
        GSM831519
        9.6%
        45.5%
        73.6%
        0.1%
        407 bp
        33.4%
        100.0%
        91.7
        5.2 X
        93.7
        4.3

        Workflow explanation

        Preprocessing of reads was done automatically with workflow tool seq2science v0.6.1. Public samples were downloaded from the Sequence Read Archive with help of the ncbi e-utilities and pysradb. Genome assembly GRCz11 was downloaded with genomepy 0.11.0. Single-end reads were trimmed with fastp v0.20.1 with default options. Paired-end reads were trimmed with fastp v0.20.1 with default options. Reads were aligned with STAR v2.7.6a with default options. Transcript abundances were quantified with Salmon v1.5.2 with options '--seqBias --gcBias --validateMappings --recoverOrphans'. Afterwards, duplicate reads were marked with Picard MarkDuplicates v2.23.8. Mapped reads were removed if they did not have a minimum mapping quality of 255, were a (secondary) multimapper or aligned inside the ENCODE blacklist.Afterwards samples were downsampled to -1 reads. General alignment statistics were collected by samtools stats v1.14. Transcript abundance estimations were aggregated and converted to gene counts using tximeta v1.10.0. Sample sequencing strandedness was inferred using RSeQC v4.0.0 in order to improve quantification accuracy. Deeptools v3.5.0 was used for the fingerprint, profile, correlation and dendrogram/heatmap plots, where the heatmap was made with options '--distanceBetweenBins 9000 --binSize 1000'. RNA-seq read duplication types were analyzed using dupRadar v1.20.0. The UCSC genome browser was used to visualize and inspect alignment. Quality control metrics were aggregated by MultiQC v1.11.

        Assembly stats

        Genome assembly GRCz11 contains of 993 contigs, with a GC-content of 36.65%, and 0.34% consists of the letter N. The N50-L50 stats are 54304671-11 and the N75-L75 stats are 48040578-18. The genome annotation contains 30954 genes.

        fastp

        fastp An ultra-fast all-in-one FASTQ preprocessor (QC, adapters, trimming, filtering, splitting...)

        Filtered Reads

        Filtering statistics of sampled reads.

        loading..

        Duplication Rates

        Duplication rates of sampled reads.

        loading..

        Insert Sizes

        Insert size estimation of sampled reads.

        loading..

        Sequence Quality

        Average sequencing quality over each base of all reads.

        loading..

        GC Content

        Average GC content over each base of all reads.

        loading..

        N content

        Average N content over each base of all reads.

        loading..

        Picard

        Picard is a set of Java command line tools for manipulating high-throughput sequencing data.

        Insert Size

        Plot shows the number of reads at a given insert size. Reads with different orientations are summed.

        loading..

        Mark Duplicates

        Number of reads, categorised by duplication state. Pair counts are doubled - see help text for details.

        The table in the Picard metrics file contains some columns referring read pairs and some referring to single reads.

        To make the numbers in this plot sum correctly, values referring to pairs are doubled according to the scheme below:

        • READS_IN_DUPLICATE_PAIRS = 2 * READ_PAIR_DUPLICATES
        • READS_IN_UNIQUE_PAIRS = 2 * (READ_PAIRS_EXAMINED - READ_PAIR_DUPLICATES)
        • READS_IN_UNIQUE_UNPAIRED = UNPAIRED_READS_EXAMINED - UNPAIRED_READ_DUPLICATES
        • READS_IN_DUPLICATE_PAIRS_OPTICAL = 2 * READ_PAIR_OPTICAL_DUPLICATES
        • READS_IN_DUPLICATE_PAIRS_NONOPTICAL = READS_IN_DUPLICATE_PAIRS - READS_IN_DUPLICATE_PAIRS_OPTICAL
        • READS_IN_DUPLICATE_UNPAIRED = UNPAIRED_READ_DUPLICATES
        • READS_UNMAPPED = UNMAPPED_READS
        loading..

        SamTools pre-sieve

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

        The pre-sieve statistics are quality metrics measured before applying (optional) minimum mapping quality, blacklist removal, mitochondrial read removal, read length filtering, and tn5 shift.

        Percent Mapped

        Alignment metrics from samtools stats; mapped vs. unmapped reads.

        For a set of samples that have come from the same multiplexed library, similar numbers of reads for each sample are expected. Large differences in numbers might indicate issues during the library preparation process. Whilst large differences in read numbers may be controlled for in downstream processings (e.g. read count normalisation), you may wish to consider whether the read depths achieved have fallen below recommended levels depending on the applications.

        Low alignment rates could indicate contamination of samples (e.g. adapter sequences), low sequencing quality or other artefacts. These can be further investigated in the sequence level QC (e.g. from FastQC).

        loading..

        Alignment metrics

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

        loading..

        deepTools

        deepTools is a suite of tools to process and analyze deep sequencing data.

        PCA plot

        PCA plot with the top two principal components calculated based on genome-wide distribution of sequence reads

        loading..

        Fingerprint plot

        Signal fingerprint according to plotFingerprint

        loading..

        Strandedness

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

        Sequencing strandedness was inferred for the following samples, and was called if 60% of the sampled reads were explained by either sense (forward) or antisense (reverse).

        Infer experiment

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

        loading..

        deepTools - Spearman correlation heatmap of reads in bins across the genome

        Spearman correlation plot generated by deeptools. Spearman correlation is a non-parametric (distribution-free) method, and assesses the monotonicity of the relationship.


        deepTools - Pearson correlation heatmap of reads in bins across the genome

        Pearson correlation plot generated by deeptools. Pearson correlation is a parametric (lots of assumptions, e.g. normality and homoscedasticity) method, and assesses the linearity of the relationship.


        dupRadar

        Figures generated by [dupRadar](https://bioconductor.riken.jp/packages/3.4/bioc/vignettes/dupRadar/inst/doc/dupRadar.html#plotting-and-interpretation). Click the link for help with interpretation.


        DESeq2 - Sample distance cluster heatmap of counts

        Euclidean distance between samples, based on variance stabilizing transformed counts (RNA: expressed genes, ChIP: bound regions, ATAC: accessible regions). Gives us an overview of similarities and dissimilarities between samples.


        DESeq2 - Spearman correlation cluster heatmap of counts

        Correlation cluster heatmap based on variance stabilizing transformed counts. Spearman correlation is a non-parametric (distribution-free) method, and assesses the monotonicity of the relationship.


        DESeq2 - Pearson correlation cluster heatmap of counts

        Correlation cluster heatmap based on variance stabilizing transformed counts. Pearson correlation is a parametric (lots of assumptions, e.g. normality and homoscedasticity) method, and assesses the linearity of the relationship.


        Samples & Config

        The samples file used for this run:

        sample assembly stage _trep descriptive_name
        GSM4079004 GRCz11 2.5hpf GSM4079004 2.5hpf_H1
        GSM4079005 GRCz11 3hpf GSM4079005 3hpf_H2
        GSM4079006 GRCz11 3.5hpf GSM4079006 3.5hpf_H3
        GSM4079007 GRCz11 4hpf GSM4079007 4hpf_H4
        GSM4079008 GRCz11 4.5hpf GSM4079008 4.5hpf_H5
        GSM4079009 GRCz11 5hpf GSM4079009 5hpf_H6
        GSM4079010 GRCz11 5.5hpf GSM4079010 5.5hpf_H7
        GSM4079011 GRCz11 2.5hpf GSM4079011 2.5hpf_H8
        GSM4079012 GRCz11 3hpf GSM4079012 3hpf_H9
        GSM4079013 GRCz11 3.5hpf GSM4079013 3.5hpf_H10
        GSM4079014 GRCz11 4hpf GSM4079014 4hpf_H11
        GSM4079015 GRCz11 4.5hpf GSM4079015 4.5hpf_H12
        GSM4079016 GRCz11 5hpf GSM4079016 5hpf_H13
        GSM4079017 GRCz11 5.5hpf GSM4079017 5.5hpf_H14
        GSM4079018 GRCz11 6hpf GSM4079018 6hpf_H15
        GSM4079019 GRCz11 2.5hpf GSM4079019 2.5hpf_H16
        GSM4079020 GRCz11 3hpf GSM4079020 3hpf_H17
        GSM4079021 GRCz11 3.5hpf GSM4079021 3.5hpf_H18
        GSM4079022 GRCz11 4hpf GSM4079022 4hpf_H19
        GSM4079023 GRCz11 4.5hpf GSM4079023 4.5hpf_H20
        GSM4079024 GRCz11 5hpf GSM4079024 5hpf_H21
        GSM4079025 GRCz11 5.5hpf GSM4079025 5.5hpf_H22
        GSM4079026 GRCz11 6hpf GSM4079026 6hpf_H23
        GSM4079027 GRCz11 2.5hpf GSM4079027 2.5hpf_H24
        GSM4079028 GRCz11 3hpf GSM4079028 3hpf_H25
        GSM4079029 GRCz11 3.5hpf GSM4079029 3.5hpf_H26
        GSM4079030 GRCz11 4hpf GSM4079030 4hpf_H27
        GSM4079031 GRCz11 4.5hpf GSM4079031 4.5hpf_H28
        GSM4079032 GRCz11 5hpf GSM4079032 5hpf_H29
        GSM4079033 GRCz11 5.5hpf GSM4079033 5.5hpf_H30
        GSM4079034 GRCz11 6hpf GSM4079034 6hpf_H31
        GSM4282070 GRCz11 2.5hpf GSM4282070 2.5hpf_H32
        GSM4282071 GRCz11 3hpf GSM4282071 3hpf_H33
        GSM4282072 GRCz11 3.5hpf GSM4282072 3.5hpf_H34
        GSM4282073 GRCz11 4hpf GSM4282073 4hpf_H35
        GSM4282074 GRCz11 4.5hpf GSM4282074 4.5hpf_H36
        GSM4282075 GRCz11 5hpf GSM4282075 5hpf_H37
        GSM4282076 GRCz11 5.5hpf GSM4282076 5.5hpf_H38
        GSM831503 GRCz11 2-4 cell GSM831503 1hpf_I1
        GSM831504 GRCz11 2-4 cell GSM831504 1hpf_I2
        GSM831505 GRCz11 1K cell GSM831505 3hpf_I3
        GSM831506 GRCz11 1K cell GSM831506 3hpf_I4
        GSM831507 GRCz11 dome GSM831507 4.3hpf_I5
        GSM831508 GRCz11 dome GSM831508 4.3hpf_I6
        GSM831509 GRCz11 shield GSM831509 6hpf_I7
        GSM831510 GRCz11 shield GSM831510 6hpf_I8
        GSM831511 GRCz11 shield GSM831511 6hpf_I9
        GSM831512 GRCz11 bud GSM831512 10hpf_I10
        GSM831513 GRCz11 bud GSM831513 10hpf_I11
        GSM831514 GRCz11 28hpf GSM831514 28hpf_I12
        GSM831515 GRCz11 28hpf GSM831515 28hpf_I13
        GSM831516 GRCz11 2dpf GSM831516 48hpf_I14
        GSM831517 GRCz11 2dpf GSM831517 48hpf_I15
        GSM831518 GRCz11 5dpf GSM831518 120hpf_I16
        GSM831519 GRCz11 5dpf GSM831519 120hpf_I17

        The config file used for this run:
        # tab-separated file of the samples
        samples: samples_pt7.tsv
        
        # pipeline file locations
        result_dir: /bank/tdewijs/results_Dre/pt7  # where to store results
        genome_dir: /bank/tdewijs/genomes  # where to look for or download the genomes
        fastq_dir: /bank/tdewijs/fastq  # where to look for or download the fastqs
        
        
        # contact info for multiqc report and trackhub
        email: tessa.dewijs2@ru.nl
        
        # produce a UCSC trackhub?
        create_trackhub: true
        
        # how to handle replicates
        technical_replicates: merge    # change to "keep" to not combine them
        
        # which trimmer to use
        trimmer: fastp
        
        # which quantifier to use
        quantifier: salmon  # or salmon or featurecounts
        
        # which aligner to use (not used for the gene counts matrix if the quantifier is Salmon)
        aligner: star
        
        # filtering after alignment (not used for the gene counts matrix if the quantifier is Salmon)
        remove_blacklist: true
        min_mapping_quality: 255  # (only keep uniquely mapped reads from STAR alignments)
        only_primary_align: true
        remove_dups: false # keep duplicates (check dupRadar in the MultiQC)
        
        ## differential gene expression analysis
        #contrasts:
        #  - 'stage_2_1'