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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_TAIR10_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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        Tool Citations

        Please remember to cite the tools that you use in your analysis.

        To help with this, you can download publication details of the tools mentioned in this report:

        About MultiQC

        This report was generated using MultiQC, version 1.14

        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 v0.9.8, 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
        September 05, 2024
        Project
        ghe_2024
        Contact E-mail
        slrinzema@science.ru.nl

        Report generated on 2024-09-05, 10:08 CEST based on data in:

        Change sample names:

        General Statistics

        Showing 4/4 rows and 14/29 columns.
        Sample Name% DuplicationM Reads After FilteringGC content% PF% AdapterInsert Size% Dups% MappedM Total seqs% Proper PairsM Total seqsGenome coverageM Genome readsM MT genome reads
        45943_Exp215_GHEtest_7_S1_Arabidopsis__RiboErase_220_A04
        15.9%
        6.6
        55.9%
        99.5%
        0.2%
        238 bp
        81.6%
        100.0%
        6.5
        100.0%
        0.5
        6.3 X
        12.8
        0.0
        45944_Exp215_GHEtest_8_S2_Arabidopsis_RiboErase_290_A09
        16.0%
        7.5
        55.2%
        99.4%
        0.3%
        223 bp
        78.8%
        100.0%
        7.4
        100.0%
        1.0
        7.0 X
        14.2
        0.0
        45945_Exp215_GHEtest_11_S1_Arabidopsis_noRibo_A27
        18.7%
        4.6
        53.0%
        99.5%
        0.3%
        202 bp
        74.8%
        100.0%
        4.5
        100.0%
        0.9
        4.0 X
        8.1
        0.0
        45946_Exp215_GHEtest_12_S2_Arabidopsis_noRibo_A31
        17.6%
        13.2
        52.4%
        99.7%
        0.5%
        187 bp
        82.6%
        100.0%
        12.9
        100.0%
        2.5
        11.6 X
        23.6
        0.0

        Workflow explanation

        Preprocessing of reads was done automatically by seq2science v0.9.8 using the alignment workflow. Paired-end reads were trimmed with fastp v0.20.1 with default options. Genome assembly hg38 was downloaded with genomepy 0.13.0. Reads were aligned with STAR v2.7.6a with default options. Afterwards, duplicate reads were marked with Picard MarkDuplicates v2.23.8. General alignment statistics were collected by samtools stats v1.14. Deeptools v3.5.1 was used for the fingerprint, profile, correlation and dendrogram/heatmap plots, where the heatmap was made with options '--distanceBetweenBins 9000 --binSize 1000'. Quality control metrics were aggregated by MultiQC v1.14.

        Assembly stats

        Genome assembly TAIR10 contains of 7 contigs, with a GC-content of 36.06%, and 0.16% consists of the letter N. The N50-L50 stats are 23459830-3 and the N75-L75 stats are 19698289-4. The genome annotation contains 31647 genes.

        fastp

        fastp An ultra-fast all-in-one FASTQ preprocessor (QC, adapters, trimming, filtering, splitting...).DOI: 10.1093/bioinformatics/bty560.

        Filtered Reads

        Filtering statistics of sampled reads.

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        Duplication Rates

        Duplication rates of sampled reads.

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        Insert Sizes

        Insert size estimation of sampled reads.

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        Sequence Quality

        Average sequencing quality over each base of all reads.

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        GC Content

        Average GC content over each base of all reads.

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        N content

        Average N content over each base of all reads.

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        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.

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        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
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        SamTools pre-sieve

        Samtools is a suite of programs for interacting with high-throughput sequencing data.DOI: 10.1093/bioinformatics/btp352.

        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).

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        Alignment metrics

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

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        SamTools post-sieve

        Samtools is a suite of programs for interacting with high-throughput sequencing data.DOI: 10.1093/bioinformatics/btp352.

        The post-sieve statistics are quality metrics measured after applying (optional) minimum mapping quality, blacklist removal, mitochondrial read removal, 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).

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        Alignment metrics

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

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        deepTools

        deepTools is a suite of tools to process and analyze deep sequencing data.DOI: 10.1093/nar/gkw257.

        PCA plot

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

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        Fingerprint plot

        Signal fingerprint according to plotFingerprint

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        Strandedness

        Strandedness package provides a number of useful modules that can comprehensively evaluate high throughput RNA-seq data.DOI: 10.1093/bioinformatics/bts356.

        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).

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        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 descriptive_name
        45937_Exp215_GHEtest_1_S1_IPSC_mRNA_A21 hg38 HomoSapiens_mRNA_1
        45938_Exp215_GHEtest_2_S2_IPSC_mRNA_A22 hg38 HomoSapiens_mRNA_2
        45939_Exp215_GHEtest_3_S1_IPSC_noRibo_A23 hg38 HomoSapiens_NoRiboerase_1
        45940_Exp215_GHEtest_4_S2_IPSC_noRibo_A24 hg38 HomoSapiens_NoRiboerase_2
        45941_Exp215_GHEtest_5_S2_IPSC_noRibo_A01 hg38 HomoSapiens_WithRiboerase_1
        45942_Exp215_GHEtest_6_S2_IPSC_noRibo_A02 hg38 HomoSapiens_WithRiboerase_2
        45943_Exp215_GHEtest_7_S1_Arabidopsis__RiboErase_220_A04 TAIR10 Arabidopsis_WithRiboerase_1
        45944_Exp215_GHEtest_8_S2_Arabidopsis_RiboErase_290_A09 TAIR10 Arabidopsis_WithRiboerase_2
        45945_Exp215_GHEtest_11_S1_Arabidopsis_noRibo_A27 TAIR10 Arabidopsis_NoRiboerase_1
        45946_Exp215_GHEtest_12_S2_Arabidopsis_noRibo_A31 TAIR10 Arabidopsis_NoRiboerase_2

        The config file used for this run: 
        # tab-separated file of the samples
samples: samples.tsv

# pipeline file locations
result_dir: ./results  # where to store results
genome_dir: /vol/slrinzema/genomes/  # where to look for or download the genomes
fastq_dir: ./samples



# contact info for multiqc report and trackhub
email: slrinzema@science.ru.nl

# produce bigwigs and a trackhub?
create_trackhub: False

# how to handle replicates
technical_replicates: merge    # change to keep to not combine them

# 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)

        MultiQC v1.14 - Written by Phil Ewels, available on GitHub.

        This report uses HighCharts, jQuery, jQuery UI, Bootstrap, FileSaver.js and clipboard.js.