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Note that additional data was saved in multiqc_hg38_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 1.9 (13f8771)

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 v0.4.3, 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.

Contact E-mail: yourmail@here.com
Workflow: joost
Date: March 08, 2021

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Report generated on 2021-03-08, 17:18 based on data in:

/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/strandedness/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.strandedness.txt
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/samtools_stats/final_bam/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.samtools-coordinate.samtools_stats.txt
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/trimming/external-HS-029RNA-U2OS-1mM-EU-1hr-18672.fastp.json
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/star/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.samtools-coordinate-unsieved.bam.mtnucratiomtnuc.json
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/plotFingerprint/hg38.tsv
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/dupRadar/hg38-dupRadar_mqc.png
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/samtools_stats/star/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.samtools-coordinate.samtools_stats.txt
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/InsertSizeMetrics/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.tsv
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/samplesconfig_mqc.html
/ceph/rimlsfnwi/data/moldevbio/heeringen/siebrenf/joost/results/qc/markdup/hg38-external-HS-029RNA-U2OS-1mM-EU-1hr-18672.samtools-coordinate.metrics.txt

Change sample names:

General Statistics

Showing ¹/₁ rows and ¹³/₂₈ columns.

Sample Name	% Duplication	% > Q30	Mb Q30 bases	GC content	% PF	% Adapter	Insert Size	Mean Insert Size	% Dups	Error rate	M Non-Primary	M Reads Mapped	% Mapped	% Proper Pairs	% MapQ 0 Reads	M Total seqs	Error rate	M Non-Primary	M Reads Mapped	% Mapped	% Proper Pairs	% MapQ 0 Reads	M Total seqs	MT genome coverage	Genome coverage	MT to Nuclear Ratio	M Genome reads	M MT genome reads
external-HS-029RNA-U2OS-1mM-EU-1hr-18672	43.5%	95.5%	114.4	45.8%	93.2%	2.4%	125 bp	140 bp	51.8%	0.00%	1.6	2.3	100.0%	100.0%	9.0%	2.3	0.00%	0.0	1.9	100.0%	100.0%	0.0%	1.9	1696.0 X	0.0 X	43139.7	3.2	0.7

Uncheck the tick box to hide columns. Click and drag the handle on the left to change order.

Sort	Group	Column	Description	ID	Scale
\|\|	fastp	% Duplication	Duplication rate in filtered reads	`pct_duplication`	None
\|\|	fastp	% > Q30	Percentage of reads > Q30 after filtering	`after_filtering_q30_rate`	None
\|\|	fastp	Mb Q30 bases	Bases > Q30 after filtering (millions)	`after_filtering_q30_bases`	base_count
\|\|	fastp	GC content	GC content after filtering	`after_filtering_gc_content`	None
\|\|	fastp	% PF	Percent reads passing filter	`pct_surviving`	None
\|\|	fastp	% Adapter	Percentage adapter-trimmed reads	`pct_adapter`	None
\|\|	Picard	Insert Size	Median Insert Size, all read orientations (bp)	`summed_median`	None
\|\|	Picard	Mean Insert Size	Mean Insert Size, all read orientations (bp)	`summed_mean`	None
\|\|	Picard	% Dups	Mark Duplicates - Percent Duplication	`PERCENT_DUPLICATION`	None
\|\|	SamTools pre-sieve	Error rate	Error rate: mismatches (NM) / bases mapped (CIGAR)	`error_rate`	None
\|\|	SamTools pre-sieve	M Non-Primary	Non-primary alignments (millions)	`non-primary_alignments`	read_count
\|\|	SamTools pre-sieve	M Reads Mapped	Reads Mapped in the bam file (millions)	`reads_mapped`	read_count
\|\|	SamTools pre-sieve	% Mapped	% Mapped Reads	`reads_mapped_percent`	None
\|\|	SamTools pre-sieve	% Proper Pairs	% Properly Paired Reads	`reads_properly_paired_percent`	None
\|\|	SamTools pre-sieve	% MapQ 0 Reads	% of Reads that are Ambiguously Placed (MapQ=0)	`reads_MQ0_percent`	None
\|\|	SamTools pre-sieve	M Total seqs	Total sequences in the bam file (millions)	`raw_total_sequences`	read_count
\|\|	SamTools post-sieve	Error rate	Error rate: mismatches (NM) / bases mapped (CIGAR)	`error_rate`	None
\|\|	SamTools post-sieve	M Non-Primary	Non-primary alignments (millions)	`non-primary_alignments`	read_count
\|\|	SamTools post-sieve	M Reads Mapped	Reads Mapped in the bam file (millions)	`reads_mapped`	read_count
\|\|	SamTools post-sieve	% Mapped	% Mapped Reads	`reads_mapped_percent`	None
\|\|	SamTools post-sieve	% Proper Pairs	% Properly Paired Reads	`reads_properly_paired_percent`	None
\|\|	SamTools post-sieve	% MapQ 0 Reads	% of Reads that are Ambiguously Placed (MapQ=0)	`reads_MQ0_percent`	None
\|\|	SamTools post-sieve	M Total seqs	Total sequences in the bam file (millions)	`raw_total_sequences`	read_count
\|\|	mtnucratio	MT genome coverage	Average coverage (X) on mitochondrial genome.	`mt_cov_avg`	None
\|\|	mtnucratio	Genome coverage	Average coverage (X) on nuclear genome.	`nuc_cov_avg`	None
\|\|	mtnucratio	MT to Nuclear Ratio	Mitochondrial to nuclear reads ratio (MTNUC)	`mt_nuc_ratio`	None
\|\|	mtnucratio	M Genome reads	Reads on the nuclear genome (millions)	`nucreads`	read_count
\|\|	mtnucratio	M MT genome reads	Reads on the mitochondrial genome (millions)	`mtreads`	read_count

fastp

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

Filtered Reads

Filtering statistics of sampled reads.

Duplication Rates

Duplication rates of sampled reads.

Insert Sizes

Insert size estimation of sampled reads.

Sequence Quality

Average sequencing quality over each base of all reads.

GC Content

Average GC content over each base of all reads.

N content

Average N content over each base of all reads.

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.

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

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).

Alignment metrics

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

SamTools post-sieve

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

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.

Alignment metrics

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

deepTools

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

Fingerprint plot

Signal fingerprint according to plotFingerprint

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).

dupRadar

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

Samples & Config

The samples file used for this run:

sample	assembly	descriptive_name
external-HS-029RNA-U2OS-1mM-EU-1hr-18672	hg38	1hr

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: ../genomes  # where to look for or download the genomes
fastq_dir: /ceph/rimlsfnwi/raw_data/fastq/2020w38-3_HGJMYBGXG/externalHansSpelbrink  # where to look for or download the fastqs


# contact info for multiqc report and trackhub
email: yourmail@here.com

# 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: htseq  # 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)
markduplicates: -Xms4G -Xmx6G MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=999  # keep duplicates (check dupRadar in the MultiQC)
remove_blacklist: true
min_mapping_quality: 255  # (only keep uniquely mapped reads from STAR alignments)
only_primary_align: true

## differential gene expression analysis
#contrasts:
#  - 'stage_2_1'

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About MultiQC

These samples were run by seq2science v0.4.3, a tool for easy preprocessing of NGS data.

General Statistics

General Statistics: Columns

fastp

Filtered Reads

Duplication Rates

Insert Sizes

Sequence Quality

GC Content

N content

Picard

Insert Size

Mark Duplicates Help

SamTools pre-sieve

Percent Mapped Help

Alignment metrics

SamTools post-sieve

Percent Mapped Help

Alignment metrics

deepTools

Fingerprint plot

Strandedness

Infer experiment

dupRadar

Samples & Config

v1.9 (13f8771)

Highlight Samples

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Mark Duplicates

Percent Mapped

Percent Mapped