MultiQC Report

A modular tool to aggregate results from bioinformatics analyses across many samples into a single report.

JavaScript Disabled

MultiQC reports use JavaScript for plots and toolbox functions. It looks like you have JavaScript disabled in your web browser. Please note that many of the report functions will not work as intended.

Report generated on 2026-08-17, 22:14 based on data in:

/projectnb/wax-dk/max/G260_G264_RNAseq/G260_RNAseq/SAMPLES
/projectnb/wax-dk/max/G260_G264_RNAseq/G260_RNAseq/Scripts/03_FASTQC
/projectnb/wax-dk/max/G260_G264_RNAseq/G260_RNAseq/Scripts/06_CollectMetrics

General Statistics

Showing ²⁰⁸/₂₀₈ rows and ¹⁵/₂₂ columns.

Sample Name	M Reads	M Reads Mapped	% Assigned	M Assigned	% rRNA	% mRNA	Insert Size	Mean Insert Size	% Dups	% Aligned	M Aligned	% Duplication	% > Q30	Mb Q30 bases	GC content	% PF	% Adapter	% Dups	% GC	Length	% Failed	M Seqs
G260-M01-STAT5a-WT_R1_001												25.7%	96.1%	3272.2	49.2%	99.2%	39.9%	67.7%	49%	150 bp	36%	12.5
G260-M01-STAT5a-WT_R2_001																		68.5%	50%	150 bp	45%	12.5
G260-M02-STAT5a-WT_R1_001												28.1%	96.3%	4406.2	49.0%	99.3%	34.7%	69.1%	49%	150 bp	36%	16.5
G260-M02-STAT5a-WT_R2_001																		69.9%	49%	150 bp	45%	16.5
G260-M03-STAT5a-WT_R1_001												22.3%	95.3%	3486.5	50.1%	99.1%	36.8%	65.4%	50%	150 bp	36%	13.3
G260-M03-STAT5a-WT_R2_001																		66.2%	51%	150 bp	45%	13.3
G260-M04-STAT5a-WT_R1_001												26.6%	95.6%	3828.5	50.5%	99.1%	22.1%	67.8%	50%	150 bp	45%	14.1
G260-M04-STAT5a-WT_R2_001																		67.8%	51%	150 bp	45%	14.1
G260-M05-STAT5a-WT_R1_001												25.1%	95.7%	3502.6	50.0%	99.2%	35.2%	66.6%	50%	150 bp	36%	13.3
G260-M05-STAT5a-WT_R2_001																		67.3%	51%	150 bp	45%	13.3
G260-M06-STAT5a-WT_R1_001												27.4%	96.3%	7681.5	49.4%	99.3%	32.0%	72.4%	49%	150 bp	36%	28.6
G260-M06-STAT5a-WT_R2_001																		71.6%	50%	150 bp	36%	28.6
G260-M07-STAT5a-WT_R1_001												28.4%	96.3%	7445.8	48.9%	99.3%	37.4%	73.8%	49%	150 bp	36%	28.0
G260-M07-STAT5a-WT_R2_001																		72.1%	49%	150 bp	36%	28.0
G260-M08-STAT5a-KO_R1_001												23.8%	96.0%	2643.1	49.2%	99.2%	27.5%	63.7%	49%	150 bp	36%	9.7
G260-M08-STAT5a-KO_R2_001																		64.4%	49%	150 bp	36%	9.7
G260-M09-STAT5a-KO_R1_001												26.6%	95.9%	4046.7	49.9%	99.2%	29.4%	66.7%	49%	150 bp	36%	15.0
G260-M09-STAT5a-KO_R2_001																		67.6%	50%	150 bp	45%	15.0
G260-M10-STAT5a-KO_R1_001												25.5%	95.8%	4568.7	49.8%	99.2%	26.4%	67.0%	49%	150 bp	36%	16.9
G260-M10-STAT5a-KO_R2_001																		67.9%	50%	150 bp	45%	16.9
G260-M11-STAT5a-KO_R1_001												29.1%	96.3%	6043.3	47.9%	99.3%	28.1%	72.0%	48%	150 bp	36%	22.2
G260-M11-STAT5a-KO_R2_001																		70.4%	48%	150 bp	36%	22.2
G260-M12-STAT5a-KO_R1_001												27.4%	96.1%	6792.9	48.5%	99.2%	35.9%	71.8%	48%	150 bp	36%	25.6
G260-M12-STAT5a-KO_R2_001																		71.1%	49%	150 bp	36%	25.6
G260-M13-STAT5a-KO_R1_001												32.1%	96.5%	7450.0	47.8%	99.3%	37.7%	75.4%	48%	150 bp	36%	27.9
G260-M13-STAT5a-KO_R2_001																		74.5%	48%	150 bp	36%	27.9
G260-M14-STAT5a-KO_R1_001												34.6%	96.0%	8121.0	51.5%	99.2%	44.2%	73.9%	51%	150 bp	36%	31.2
G260-M14-STAT5a-KO_R2_001																		72.6%	52%	150 bp	36%	31.2
G260-M15-STAT5a-WT_R1_001												17.3%	96.1%	1958.6	49.7%	99.2%	27.8%	57.4%	49%	150 bp	36%	7.3
G260-M15-STAT5a-WT_R2_001																		58.5%	50%	150 bp	45%	7.3
G260-M16-STAT5a-WT_R1_001												25.5%	96.0%	6216.8	49.2%	99.2%	35.7%	69.7%	49%	150 bp	36%	23.3
G260-M16-STAT5a-WT_R2_001																		68.8%	50%	150 bp	45%	23.3
G260-M17-STAT5a-WT_R1_001												22.1%	95.4%	6322.6	50.1%	99.1%	36.7%	67.0%	50%	150 bp	45%	24.1
G260-M17-STAT5a-WT_R2_001																		67.0%	51%	150 bp	45%	24.1
G260-M18-STAT5a-WT_R1_001												27.6%	95.6%	5841.1	50.8%	99.2%	40.3%	69.6%	50%	150 bp	45%	22.6
G260-M18-STAT5a-WT_R2_001																		69.8%	52%	150 bp	45%	22.6
G260-M19-STAT5a-WT_R1_001												27.3%	96.3%	7579.1	48.6%	99.3%	31.0%	71.8%	48%	150 bp	36%	28.3
G260-M19-STAT5a-WT_R2_001																		70.5%	49%	150 bp	36%	28.3
G260-M20-STAT5a-WT_R1_001												26.0%	96.3%	9103.6	49.3%	99.3%	40.1%	73.6%	49%	150 bp	36%	34.5
G260-M20-STAT5a-WT_R2_001																		72.3%	50%	150 bp	36%	34.5
G260-M21-STAT5a-WT_R1_001												27.9%	96.4%	9039.1	49.1%	99.3%	35.6%	73.2%	49%	150 bp	36%	33.7
G260-M21-STAT5a-WT_R2_001																		72.1%	49%	150 bp	36%	33.7
G260-M22-STAT5a-KO_R1_001												21.8%	96.1%	3122.7	49.4%	99.2%	23.3%	62.1%	49%	150 bp	36%	11.4
G260-M22-STAT5a-KO_R2_001																		63.3%	50%	150 bp	36%	11.4
G260-M23-STAT5a-KO_R1_001												19.9%	96.2%	2609.9	49.1%	99.2%	34.4%	60.7%	49%	150 bp	36%	9.8
G260-M23-STAT5a-KO_R2_001																		61.6%	49%	150 bp	36%	9.8
G260-M24-STAT5a-KO_R1_001												23.7%	96.1%	3416.8	49.4%	99.2%	37.5%	64.9%	49%	150 bp	36%	13.0
G260-M24-STAT5a-KO_R2_001																		65.6%	50%	150 bp	36%	13.0
G260-M25-STAT5a-KO_R1_001												28.1%	96.1%	8851.1	51.1%	99.2%	33.3%	72.3%	51%	150 bp	36%	33.2
G260-M25-STAT5a-KO_R2_001																		71.9%	51%	150 bp	36%	33.2
G260-M26-STAT5a-KO_R1_001												25.5%	96.3%	8219.6	49.2%	99.3%	42.1%	71.0%	49%	150 bp	36%	31.3
G260-M26-STAT5a-KO_R2_001																		70.1%	49%	150 bp	36%	31.3
G260_M01_G260_M01										63.4%	7.9
G260_M01_primary_unique					0.7%	86.0%	297 bp	757 bp	29.7%
G260_M01_sorted			92.2%	7.3
G260_M01_statistics_for_all_accepted_reads	20.3	20.3
G260_M01_statistics_for_primary_reads	17.4	17.4
G260_M01_statistics_for_primary_unique_reads	15.9	15.9
G260_M02_G260_M02										70.1%	11.6
G260_M02_primary_unique					0.5%	86.2%	342 bp	872 bp	34.4%
G260_M02_sorted			92.2%	10.7
G260_M02_statistics_for_all_accepted_reads	28.8	28.8
G260_M02_statistics_for_primary_reads	25.2	25.2
G260_M02_statistics_for_primary_unique_reads	23.2	23.2
G260_M03_G260_M03										66.0%	8.8
G260_M03_primary_unique					0.3%	86.1%	304 bp	764 bp	26.3%
G260_M03_sorted			92.9%	8.2
G260_M03_statistics_for_all_accepted_reads	21.8	21.8
G260_M03_statistics_for_primary_reads	19.0	19.0
G260_M03_statistics_for_primary_unique_reads	17.6	17.6
G260_M04_G260_M04										73.7%	10.4
G260_M04_primary_unique					0.3%	87.0%	416 bp	988 bp	30.2%
G260_M04_sorted			93.5%	9.7
G260_M04_statistics_for_all_accepted_reads	24.8	24.8
G260_M04_statistics_for_primary_reads	22.2	22.2
G260_M04_statistics_for_primary_unique_reads	20.7	20.7
G260_M05_G260_M05										67.4%	8.9
G260_M05_primary_unique					0.5%	86.1%	318 bp	799 bp	30.3%
G260_M05_sorted			92.8%	8.3
G260_M05_statistics_for_all_accepted_reads	22.1	22.1
G260_M05_statistics_for_primary_reads	19.4	19.4
G260_M05_statistics_for_primary_unique_reads	17.9	17.9
G260_M06_G260_M06										76.0%	21.7
G260_M06_primary_unique					0.2%	87.4%	407 bp	959 bp	34.6%
G260_M06_sorted			93.8%	20.4
G260_M06_statistics_for_all_accepted_reads	52.0	52.0
G260_M06_statistics_for_primary_reads	46.5	46.5
G260_M06_statistics_for_primary_unique_reads	43.5	43.5
G260_M07_G260_M07										67.5%	18.9
G260_M07_primary_unique					0.4%	86.7%	326 bp	851 bp	31.1%
G260_M07_sorted			93.5%	17.7
G260_M07_statistics_for_all_accepted_reads	47.3	47.3
G260_M07_statistics_for_primary_reads	41.0	41.0
G260_M07_statistics_for_primary_unique_reads	37.8	37.8
G260_M08_G260_M08										74.5%	7.2
G260_M08_primary_unique					0.3%	86.5%	355 bp	879 bp	29.5%
G260_M08_sorted			93.0%	6.7
G260_M08_statistics_for_all_accepted_reads	17.6	17.6
G260_M08_statistics_for_primary_reads	15.6	15.6
G260_M08_statistics_for_primary_unique_reads	14.5	14.5
G260_M09_G260_M09										72.0%	10.8
G260_M09_primary_unique					0.3%	86.4%	344 bp	863 bp	31.5%
G260_M09_sorted			93.3%	10.1
G260_M09_statistics_for_all_accepted_reads	26.3	26.3
G260_M09_statistics_for_primary_reads	23.3	23.3
G260_M09_statistics_for_primary_unique_reads	21.7	21.7
G260_M10_G260_M10										72.3%	12.2
G260_M10_primary_unique					0.5%	85.8%	377 bp	935 bp	29.8%
G260_M10_sorted			93.1%	11.4
G260_M10_statistics_for_all_accepted_reads	29.8	29.8
G260_M10_statistics_for_primary_reads	26.3	26.3
G260_M10_statistics_for_primary_unique_reads	24.4	24.4
G260_M11_G260_M11										68.9%	15.3
G260_M11_primary_unique					0.3%	82.8%	303 bp	750 bp	29.6%
G260_M11_sorted			91.5%	14.0
G260_M11_statistics_for_all_accepted_reads	38.5	38.5
G260_M11_statistics_for_primary_reads	33.3	33.3
G260_M11_statistics_for_primary_unique_reads	30.6	30.6
G260_M12_G260_M12										66.4%	17.0
G260_M12_primary_unique					0.2%	84.6%	311 bp	793 bp	29.5%
G260_M12_sorted			92.1%	15.6
G260_M12_statistics_for_all_accepted_reads	42.8	42.8
G260_M12_statistics_for_primary_reads	37.1	37.1
G260_M12_statistics_for_primary_unique_reads	34.0	34.0
G260_M13_G260_M13										63.6%	17.8
G260_M13_primary_unique					0.5%	86.0%	292 bp	782 bp	32.5%
G260_M13_sorted			91.9%	16.3
G260_M13_statistics_for_all_accepted_reads	47.1	47.1
G260_M13_statistics_for_primary_reads	39.4	39.4
G260_M13_statistics_for_primary_unique_reads	35.5	35.5
G260_M14_G260_M14										65.0%	20.3
G260_M14_primary_unique					20.2%	62.2%	183 bp	276 bp	42.6%
G260_M14_sorted			81.4%	16.5
G260_M14_statistics_for_all_accepted_reads	50.3	50.3
G260_M14_statistics_for_primary_reads	43.9	43.9
G260_M14_statistics_for_primary_unique_reads	40.5	40.5
G260_M15_G260_M15										75.4%	5.5
G260_M15_primary_unique					0.5%	86.7%	363 bp	912 bp	22.1%
G260_M15_sorted			93.8%	5.1
G260_M15_statistics_for_all_accepted_reads	12.9	12.9
G260_M15_statistics_for_primary_reads	11.7	11.7
G260_M15_statistics_for_primary_unique_reads	10.9	10.9
G260_M16_G260_M16										74.1%	17.3
G260_M16_primary_unique					0.2%	84.8%	315 bp	818 bp	32.5%
G260_M16_sorted			92.3%	16.0
G260_M16_statistics_for_all_accepted_reads	41.1	41.1
G260_M16_statistics_for_primary_reads	37.2	37.2
G260_M16_statistics_for_primary_unique_reads	34.6	34.6
G260_M17_G260_M17										72.3%	17.4
G260_M17_primary_unique					0.2%	86.6%	285 bp	721 bp	29.3%
G260_M17_sorted			93.6%	16.3
G260_M17_statistics_for_all_accepted_reads	40.4	40.4
G260_M17_statistics_for_primary_reads	37.0	37.0
G260_M17_statistics_for_primary_unique_reads	34.8	34.8
G260_M18_G260_M18										66.3%	15.0
G260_M18_primary_unique					0.3%	85.2%	283 bp	723 bp	33.1%
G260_M18_sorted			92.8%	13.9
G260_M18_statistics_for_all_accepted_reads	35.8	35.8
G260_M18_statistics_for_primary_reads	32.1	32.1
G260_M18_statistics_for_primary_unique_reads	29.9	29.9
G260_M19_G260_M19										74.9%	21.2
G260_M19_primary_unique					0.2%	84.6%	320 bp	818 bp	35.0%
G260_M19_sorted			92.9%	19.7
G260_M19_statistics_for_all_accepted_reads	50.7	50.7
G260_M19_statistics_for_primary_reads	45.7	45.7
G260_M19_statistics_for_primary_unique_reads	42.4	42.4
G260_M20_G260_M20										72.3%	25.0
G260_M20_primary_unique					0.4%	87.2%	291 bp	770 bp	32.7%
G260_M20_sorted			93.1%	23.2
G260_M20_statistics_for_all_accepted_reads	59.8	59.8
G260_M20_statistics_for_primary_reads	53.8	53.8
G260_M20_statistics_for_primary_unique_reads	49.9	49.9
G260_M21_G260_M21										76.5%	25.8
G260_M21_primary_unique					0.3%	86.4%	293 bp	768 bp	35.3%
G260_M21_sorted			92.9%	23.9
G260_M21_statistics_for_all_accepted_reads	61.0	61.0
G260_M21_statistics_for_primary_reads	55.3	55.3
G260_M21_statistics_for_primary_unique_reads	51.5	51.5
G260_M22_G260_M22										79.1%	9.0
G260_M22_primary_unique					0.4%	86.9%	403 bp	991 bp	28.6%
G260_M22_sorted			93.6%	8.4
G260_M22_statistics_for_all_accepted_reads	21.4	21.4
G260_M22_statistics_for_primary_reads	19.3	19.3
G260_M22_statistics_for_primary_unique_reads	18.0	18.0
G260_M23_G260_M23										72.9%	7.1
G260_M23_primary_unique					0.4%	85.8%	313 bp	787 bp	25.9%
G260_M23_sorted			93.0%	6.6
G260_M23_statistics_for_all_accepted_reads	17.2	17.2
G260_M23_statistics_for_primary_reads	15.3	15.3
G260_M23_statistics_for_primary_unique_reads	14.3	14.3
G260_M24_G260_M24										71.0%	9.2
G260_M24_primary_unique					0.3%	86.1%	309 bp	789 bp	31.4%
G260_M24_sorted			93.1%	8.6
G260_M24_statistics_for_all_accepted_reads	22.2	22.2
G260_M24_statistics_for_primary_reads	19.8	19.8
G260_M24_statistics_for_primary_unique_reads	18.4	18.4
G260_M25_G260_M25										73.4%	24.4
G260_M25_primary_unique					11.5%	75.0%	226 bp	464 bp	36.9%
G260_M25_sorted			87.2%	21.3
G260_M25_statistics_for_all_accepted_reads	57.3	57.3
G260_M25_statistics_for_primary_reads	52.0	52.0
G260_M25_statistics_for_primary_unique_reads	48.7	48.7
G260_M26_G260_M26										73.1%	22.9
G260_M26_primary_unique					0.3%	86.2%	299 bp	790 bp	33.2%
G260_M26_sorted			93.2%	21.3
G260_M26_statistics_for_all_accepted_reads	54.3	54.3
G260_M26_statistics_for_primary_reads	49.0	49.0
G260_M26_statistics_for_primary_unique_reads	45.7	45.7

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

Sort	Group	Column	Description	ID	Scale
\|\|	Samtools	M Reads	Total reads in the bam file (millions)	`flagstat_total`	read_count
\|\|	Samtools	M Reads Mapped	Reads Mapped in the bam file (millions)	`mapped_passed`	read_count
\|\|	featureCounts	% Assigned	% Assigned reads	`percent_assigned`	None
\|\|	featureCounts	M Assigned	Assigned reads (millions)	`Assigned`	read_count
\|\|	Picard	% rRNA	Percent of aligned bases overlapping ribosomal RNA regions	`PCT_RIBOSOMAL_BASES`	None
\|\|	Picard	% mRNA	Percent of aligned bases overlapping UTRs and coding regions of mRNA transcripts	`PCT_MRNA_BASES`	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
\|\|	STAR	% Aligned	% Uniquely mapped reads	`uniquely_mapped_percent`	None
\|\|	STAR	M Aligned	Uniquely mapped reads (millions)	`uniquely_mapped`	read_count
\|\|	fastp	% Duplication	Duplication rate before filtering	`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
\|\|	FastQC	% Dups	% Duplicate Reads	`percent_duplicates`	None
\|\|	FastQC	% GC	Average % GC Content	`percent_gc`	None
\|\|	FastQC	Length	Average Sequence Length (bp)	`avg_sequence_length`	None
\|\|	FastQC	% Failed	Percentage of modules failed in FastQC report (includes those not plotted here)	`percent_fails`	None
\|\|	FastQC	M Seqs	Total Sequences (millions)	`total_sequences`	read_count

RSeQC

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

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).

featureCounts

Subread featureCounts is a highly efficient general-purpose read summarization program that counts mapped reads for genomic features such as genes, exons, promoter, gene bodies, genomic bins and chromosomal locations.

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

RnaSeqMetrics Assignment

Number of bases in primary alignments that align to regions in the reference genome.

RnaSeqMetrics Strand Mapping

Number of aligned reads that map to the correct strand.

Gene Coverage

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.

STAR

STAR is an ultrafast universal RNA-seq aligner.

Alignment Scores

Gene Counts

Statistics from results generated using --quantMode GeneCounts. The three tabs show counts for unstranded RNA-seq, counts for the 1st read strand aligned with RNA and counts for the 2nd read strand aligned with RNA.

fastp

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

Filtered Reads

Filtering statistics 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.

FastQC

FastQC is a quality control tool for high throughput sequence data, written by Simon Andrews at the Babraham Institute in Cambridge.

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.

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.

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.

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.

Sequence Length Distribution

All samples have sequences of a single length (150bp).

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.

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.

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.

Flat image plot. Toolbox functions such as highlighting / hiding samples will not work (see the docs).

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.

Min:

Max:

Toggle navigation v1.11

MultiQC Toolbox

Apply Highlight Samples

Apply Rename Samples

Apply Show / Hide Samples

Export Plots

Choose Plots

Save Settings

Load Settings

About MultiQC

General Statistics

General Statistics: Columns

RSeQC

Infer experiment

featureCounts

Picard

Insert Size

Mark Duplicates Help

RnaSeqMetrics Assignment

RnaSeqMetrics Strand Mapping

Gene Coverage

Samtools

Samtools Flagstat

STAR

Alignment Scores

Gene Counts

fastp

Filtered Reads

Insert Sizes

Sequence Quality

GC Content

N content

FastQC

Sequence Quality Histograms Help

Per Sequence Quality Scores Help

Per Base Sequence Content Help

Rollover for sample name

Per Sequence GC Content Help

Per Base N Content Help

Sequence Length Distribution

Sequence Duplication Levels Help

Overrepresented sequences Help

Adapter Content Help

Status Checks Help

v1.11

Highlight Samples

Rename Samples

Show / Hide Samples

Mark Duplicates

Sequence Quality Histograms

Per Sequence Quality Scores

Per Base Sequence Content

Per Sequence GC Content

Per Base N Content

Sequence Duplication Levels

Overrepresented sequences

Adapter Content

Status Checks