MultiQC Report

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

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Report generated on 2026-02-28, 19:45 based on data in:

/projectnb/wax-dk/max/G253_RNASEQ_DevinKenney/SAMPLES
/projectnb/wax-dk/max/G253_RNASEQ_DevinKenney/Scripts/03_FASTQC
/projectnb/wax-dk/max/G253_RNASEQ_DevinKenney/Scripts/06_CollectMetrics

General Statistics

Showing ²⁵⁶/₂₅₆ 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
31_R1_001												18.6%	92.5%	5202.6	50.4%	98.2%	8.2%	53.6%	50%	150 bp	27%	19.4
31_R2_001																		52.3%	51%	150 bp	27%	19.4
32_R1_001												16.7%	91.9%	5813.4	50.9%	97.9%	9.2%	54.0%	51%	150 bp	27%	21.9
32_R2_001																		52.3%	51%	150 bp	27%	21.9
33_R1_001												17.2%	91.8%	5889.4	50.5%	97.7%	9.6%	52.9%	50%	150 bp	27%	22.3
33_R2_001																		51.4%	51%	150 bp	27%	22.3
34_R1_001												18.0%	91.5%	6265.6	50.8%	97.7%	8.7%	55.9%	50%	150 bp	27%	23.7
34_R2_001																		54.0%	51%	150 bp	27%	23.7
35_R1_001												16.6%	91.3%	6069.8	50.5%	97.7%	7.4%	53.4%	50%	150 bp	27%	23.0
35_R2_001																		51.1%	51%	150 bp	27%	23.0
36_R1_001												17.0%	91.5%	7087.7	50.9%	97.7%	9.1%	55.3%	51%	150 bp	27%	26.8
36_R2_001																		53.6%	51%	150 bp	27%	26.8
37_R1_001												18.0%	92.5%	6464.3	50.6%	98.2%	8.6%	53.1%	50%	150 bp	27%	24.1
37_R2_001																		51.8%	51%	150 bp	27%	24.1
38_R1_001												17.9%	91.6%	5652.5	50.5%	97.8%	8.2%	57.1%	50%	150 bp	27%	21.3
38_R2_001																		55.3%	51%	150 bp	27%	21.3
39_R1_001												18.5%	92.3%	5327.5	50.6%	98.2%	8.5%	55.3%	50%	150 bp	27%	19.9
39_R2_001																		53.7%	51%	150 bp	27%	19.9
40_R1_001												17.5%	91.9%	5460.6	51.6%	97.8%	10.4%	57.3%	51%	150 bp	27%	20.6
40_R2_001																		55.3%	52%	150 bp	27%	20.6
41_R1_001												17.6%	91.5%	6142.8	50.7%	97.6%	8.0%	55.1%	50%	150 bp	27%	23.2
41_R2_001																		53.3%	51%	150 bp	27%	23.2
42_R1_001												19.3%	91.6%	7096.8	50.5%	97.8%	8.7%	59.8%	50%	150 bp	27%	26.8
42_R2_001																		57.6%	51%	150 bp	27%	26.8
43_R1_001												16.9%	91.6%	4311.9	50.2%	97.4%	6.6%	50.6%	50%	150 bp	27%	16.3
43_R2_001																		48.6%	51%	150 bp	27%	16.3
44_R1_001												18.6%	92.2%	5181.0	50.6%	97.5%	7.7%	55.6%	50%	150 bp	27%	19.5
44_R2_001																		54.0%	51%	150 bp	36%	19.5
45_R1_001												18.4%	93.0%	5003.7	50.4%	98.1%	7.2%	53.5%	50%	150 bp	27%	18.5
45_R2_001																		52.1%	51%	150 bp	36%	18.5
46_R1_001												19.8%	92.7%	4733.1	51.1%	97.8%	8.6%	57.9%	51%	150 bp	27%	17.7
46_R2_001																		56.1%	51%	150 bp	36%	17.7
47_R1_001												19.1%	93.2%	5316.4	50.6%	98.2%	8.4%	55.3%	50%	150 bp	27%	19.7
47_R2_001																		54.2%	51%	150 bp	27%	19.7
48_R1_001												17.9%	92.3%	5174.0	50.4%	97.7%	7.2%	56.2%	50%	150 bp	27%	19.4
48_R2_001																		54.2%	51%	150 bp	36%	19.4
49_R1_001												17.0%	92.2%	5063.4	50.7%	97.5%	7.9%	53.7%	50%	150 bp	27%	19.0
49_R2_001																		52.0%	51%	150 bp	36%	19.0
50_R1_001												16.4%	91.1%	4432.9	50.3%	97.0%	6.3%	51.3%	50%	150 bp	27%	16.9
50_R2_001																		49.3%	51%	150 bp	27%	16.9
51_R1_001												16.3%	91.1%	5254.7	50.7%	97.5%	7.2%	53.8%	50%	150 bp	27%	20.0
51_R2_001																		52.0%	51%	150 bp	27%	20.0
52_R1_001												16.7%	91.5%	5672.6	50.2%	97.6%	9.3%	55.4%	50%	150 bp	27%	21.5
52_R2_001																		53.8%	50%	150 bp	27%	21.5
53_R1_001												18.1%	92.5%	5627.0	50.2%	98.3%	8.2%	55.4%	50%	150 bp	27%	20.9
53_R2_001																		54.2%	50%	150 bp	27%	20.9
54_R1_001												35.2%	92.8%	6310.0	49.1%	98.2%	12.8%	79.0%	49%	150 bp	36%	23.6
54_R2_001																		78.0%	49%	150 bp	36%	23.6
55_R1_001												17.5%	92.2%	4729.2	49.9%	98.1%	8.2%	52.5%	50%	150 bp	27%	17.7
55_R2_001																		51.4%	50%	150 bp	27%	17.7
56_R1_001												15.9%	91.3%	5832.5	50.4%	97.6%	8.3%	52.5%	50%	150 bp	27%	22.1
56_R2_001																		51.0%	51%	150 bp	27%	22.1
57_R1_001												16.3%	91.0%	5857.4	50.6%	97.2%	7.3%	53.2%	50%	150 bp	27%	22.4
57_R2_001																		51.8%	51%	150 bp	36%	22.4
58_R1_001												15.6%	91.2%	5370.1	50.7%	97.4%	8.8%	51.3%	50%	150 bp	27%	20.5
58_R2_001																		50.2%	51%	150 bp	36%	20.5
59_R1_001												15.4%	91.0%	6008.2	50.8%	97.6%	9.0%	52.8%	51%	150 bp	27%	22.9
59_R2_001																		50.9%	51%	150 bp	27%	22.9
60_R1_001												16.5%	91.0%	5613.8	50.9%	97.5%	7.9%	53.8%	51%	150 bp	27%	21.4
60_R2_001																		51.9%	51%	150 bp	27%	21.4
61_R1_001												16.3%	92.0%	5897.4	50.5%	98.1%	9.4%	51.3%	50%	150 bp	27%	22.1
61_R2_001																		49.7%	51%	150 bp	18%	22.1
62_R1_001												15.7%	90.6%	6421.9	50.4%	97.4%	7.0%	53.7%	50%	150 bp	27%	24.6
62_R2_001																		51.6%	51%	150 bp	36%	24.6
G253_M31_G253_M31										88.7%	17.2
G253_M31_primary_unique					2.2%	88.4%	511 bp	1193 bp	40.6%
G253_M31_sorted			96.0%	16.5
G253_M31_statistics_for_all_accepted_reads	37.8	37.8
G253_M31_statistics_for_primary_reads	35.8	35.8
G253_M31_statistics_for_primary_unique_reads	34.4	34.4
G253_M32_G253_M32										88.7%	19.4
G253_M32_primary_unique					1.6%	89.5%	532 bp	1269 bp	39.2%
G253_M32_sorted			96.2%	18.7
G253_M32_statistics_for_all_accepted_reads	42.4	42.4
G253_M32_statistics_for_primary_reads	40.3	40.3
G253_M32_statistics_for_primary_unique_reads	38.8	38.8
G253_M33_G253_M33										88.5%	19.7
G253_M33_primary_unique					1.4%	87.6%	502 bp	1153 bp	40.6%
G253_M33_sorted			95.6%	18.8
G253_M33_statistics_for_all_accepted_reads	42.8	42.8
G253_M33_statistics_for_primary_reads	40.8	40.8
G253_M33_statistics_for_primary_unique_reads	39.4	39.4
G253_M34_G253_M34										88.8%	21.1
G253_M34_primary_unique					1.6%	89.9%	586 bp	1393 bp	43.4%
G253_M34_sorted			96.6%	20.4
G253_M34_statistics_for_all_accepted_reads	45.7	45.7
G253_M34_statistics_for_primary_reads	43.6	43.6
G253_M34_statistics_for_primary_unique_reads	42.1	42.1
G253_M35_G253_M35										88.9%	20.4
G253_M35_primary_unique					1.9%	88.1%	551 bp	1297 bp	40.8%
G253_M35_sorted			95.9%	19.6
G253_M35_statistics_for_all_accepted_reads	44.3	44.3
G253_M35_statistics_for_primary_reads	42.3	42.3
G253_M35_statistics_for_primary_unique_reads	40.9	40.9
G253_M36_G253_M36										88.6%	23.8
G253_M36_primary_unique					1.7%	89.4%	541 bp	1278 bp	41.4%
G253_M36_sorted			96.6%	23.0
G253_M36_statistics_for_all_accepted_reads	51.2	51.2
G253_M36_statistics_for_primary_reads	49.1	49.1
G253_M36_statistics_for_primary_unique_reads	47.6	47.6
G253_M37_G253_M37										89.6%	21.6
G253_M37_primary_unique					1.4%	85.1%	495 bp	1123 bp	41.2%
G253_M37_sorted			94.0%	20.3
G253_M37_statistics_for_all_accepted_reads	46.7	46.7
G253_M37_statistics_for_primary_reads	44.6	44.6
G253_M37_statistics_for_primary_unique_reads	43.1	43.1
G253_M38_G253_M38										87.7%	18.7
G253_M38_primary_unique					3.3%	87.0%	468 bp	1002 bp	43.7%
G253_M38_sorted			95.8%	17.9
G253_M38_statistics_for_all_accepted_reads	41.3	41.3
G253_M38_statistics_for_primary_reads	39.0	39.0
G253_M38_statistics_for_primary_unique_reads	37.4	37.4
G253_M39_G253_M39										88.9%	17.7
G253_M39_primary_unique					2.2%	89.1%	537 bp	1251 bp	41.6%
G253_M39_sorted			96.0%	17.0
G253_M39_statistics_for_all_accepted_reads	38.6	38.6
G253_M39_statistics_for_primary_reads	36.7	36.7
G253_M39_statistics_for_primary_unique_reads	35.3	35.3
G253_M40_G253_M40										87.0%	17.9
G253_M40_primary_unique					2.8%	89.5%	571 bp	1349 bp	41.5%
G253_M40_sorted			96.2%	17.3
G253_M40_statistics_for_all_accepted_reads	39.6	39.6
G253_M40_statistics_for_primary_reads	37.4	37.4
G253_M40_statistics_for_primary_unique_reads	35.9	35.9
G253_M41_G253_M41										88.8%	20.6
G253_M41_primary_unique					1.8%	89.2%	563 bp	1351 bp	42.3%
G253_M41_sorted			96.4%	19.9
G253_M41_statistics_for_all_accepted_reads	44.8	44.8
G253_M41_statistics_for_primary_reads	42.8	42.8
G253_M41_statistics_for_primary_unique_reads	41.3	41.3
G253_M42_G253_M42										87.5%	23.5
G253_M42_primary_unique					2.4%	88.0%	605 bp	1437 bp	45.2%
G253_M42_sorted			94.8%	22.2
G253_M42_statistics_for_all_accepted_reads	52.2	52.2
G253_M42_statistics_for_primary_reads	49.2	49.2
G253_M42_statistics_for_primary_unique_reads	46.9	46.9
G253_M43_G253_M43										88.4%	14.4
G253_M43_primary_unique					2.0%	88.3%	581 bp	1379 bp	39.3%
G253_M43_sorted			95.8%	13.8
G253_M43_statistics_for_all_accepted_reads	31.4	31.4
G253_M43_statistics_for_primary_reads	29.9	29.9
G253_M43_statistics_for_primary_unique_reads	28.8	28.8
G253_M44_G253_M44										86.8%	16.9
G253_M44_primary_unique					2.8%	88.0%	592 bp	1428 bp	40.6%
G253_M44_sorted			95.2%	16.1
G253_M44_statistics_for_all_accepted_reads	37.7	37.7
G253_M44_statistics_for_primary_reads	35.5	35.5
G253_M44_statistics_for_primary_unique_reads	33.8	33.8
G253_M45_G253_M45										89.1%	16.5
G253_M45_primary_unique					2.4%	87.8%	566 bp	1355 bp	38.6%
G253_M45_sorted			96.1%	15.8
G253_M45_statistics_for_all_accepted_reads	35.9	35.9
G253_M45_statistics_for_primary_reads	34.2	34.2
G253_M45_statistics_for_primary_unique_reads	33.0	33.0
G253_M46_G253_M46										87.5%	15.5
G253_M46_primary_unique					2.4%	90.2%	648 bp	1505 bp	42.3%
G253_M46_sorted			96.4%	14.9
G253_M46_statistics_for_all_accepted_reads	34.1	34.1
G253_M46_statistics_for_primary_reads	32.3	32.3
G253_M46_statistics_for_primary_unique_reads	30.9	30.9
G253_M47_G253_M47										89.0%	17.5
G253_M47_primary_unique					1.7%	89.9%	570 bp	1340 bp	39.5%
G253_M47_sorted			96.6%	16.9
G253_M47_statistics_for_all_accepted_reads	38.2	38.2
G253_M47_statistics_for_primary_reads	36.3	36.3
G253_M47_statistics_for_primary_unique_reads	35.0	35.0
G253_M48_G253_M48										87.4%	16.9
G253_M48_primary_unique					2.4%	88.0%	596 bp	1384 bp	39.8%
G253_M48_sorted			95.6%	16.2
G253_M48_statistics_for_all_accepted_reads	37.8	37.8
G253_M48_statistics_for_primary_reads	35.5	35.5
G253_M48_statistics_for_primary_unique_reads	33.9	33.9
G253_M49_G253_M49										88.5%	16.9
G253_M49_primary_unique					1.3%	91.0%	627 bp	1490 bp	38.3%
G253_M49_sorted			97.0%	16.3
G253_M49_statistics_for_all_accepted_reads	36.5	36.5
G253_M49_statistics_for_primary_reads	34.9	34.9
G253_M49_statistics_for_primary_unique_reads	33.7	33.7
G253_M50_G253_M50										88.1%	14.9
G253_M50_primary_unique					2.0%	89.7%	631 bp	1523 bp	39.6%
G253_M50_sorted			96.6%	14.4
G253_M50_statistics_for_all_accepted_reads	32.3	32.3
G253_M50_statistics_for_primary_reads	30.9	30.9
G253_M50_statistics_for_primary_unique_reads	29.8	29.8
G253_M51_G253_M51										88.8%	17.7
G253_M51_primary_unique					1.9%	89.5%	577 bp	1350 bp	40.9%
G253_M51_sorted			96.4%	17.1
G253_M51_statistics_for_all_accepted_reads	38.4	38.4
G253_M51_statistics_for_primary_reads	36.7	36.7
G253_M51_statistics_for_primary_unique_reads	35.5	35.5
G253_M52_G253_M52										87.6%	18.8
G253_M52_primary_unique					1.6%	88.4%	472 bp	1021 bp	41.0%
G253_M52_sorted			95.3%	18.0
G253_M52_statistics_for_all_accepted_reads	41.5	41.5
G253_M52_statistics_for_primary_reads	39.3	39.3
G253_M52_statistics_for_primary_unique_reads	37.7	37.7
G253_M53_G253_M53										88.4%	18.5
G253_M53_primary_unique					1.9%	88.3%	492 bp	1116 bp	40.3%
G253_M53_sorted			95.3%	17.6
G253_M53_statistics_for_all_accepted_reads	41.3	41.3
G253_M53_statistics_for_primary_reads	38.8	38.8
G253_M53_statistics_for_primary_unique_reads	37.0	37.0
G253_M54_G253_M54										80.1%	18.9
G253_M54_primary_unique					5.0%	83.7%	309 bp	527 bp	78.7%
G253_M54_sorted			95.5%	18.0
G253_M54_statistics_for_all_accepted_reads	48.1	48.1
G253_M54_statistics_for_primary_reads	41.8	41.8
G253_M54_statistics_for_primary_unique_reads	37.8	37.8
G253_M55_G253_M55										88.5%	15.7
G253_M55_primary_unique					1.6%	86.9%	470 bp	1011 bp	40.5%
G253_M55_sorted			94.6%	14.8
G253_M55_statistics_for_all_accepted_reads	34.6	34.6
G253_M55_statistics_for_primary_reads	32.7	32.7
G253_M55_statistics_for_primary_unique_reads	31.3	31.3
G253_M56_G253_M56										88.6%	19.6
G253_M56_primary_unique					1.3%	86.9%	507 bp	1141 bp	40.2%
G253_M56_sorted			94.7%	18.6
G253_M56_statistics_for_all_accepted_reads	42.5	42.5
G253_M56_statistics_for_primary_reads	40.6	40.6
G253_M56_statistics_for_primary_unique_reads	39.2	39.2
G253_M57_G253_M57										88.5%	19.8
G253_M57_primary_unique					1.4%	89.1%	545 bp	1263 bp	41.3%
G253_M57_sorted			96.2%	19.0
G253_M57_statistics_for_all_accepted_reads	43.1	43.1
G253_M57_statistics_for_primary_reads	41.0	41.0
G253_M57_statistics_for_primary_unique_reads	39.6	39.6
G253_M58_G253_M58										88.6%	18.1
G253_M58_primary_unique					1.0%	88.7%	536 bp	1231 bp	39.8%
G253_M58_sorted			95.9%	17.4
G253_M58_statistics_for_all_accepted_reads	39.1	39.1
G253_M58_statistics_for_primary_reads	37.4	37.4
G253_M58_statistics_for_primary_unique_reads	36.3	36.3
G253_M59_G253_M59										88.2%	20.2
G253_M59_primary_unique					1.2%	87.7%	537 bp	1252 bp	39.5%
G253_M59_sorted			95.0%	19.2
G253_M59_statistics_for_all_accepted_reads	44.1	44.1
G253_M59_statistics_for_primary_reads	41.9	41.9
G253_M59_statistics_for_primary_unique_reads	40.4	40.4
G253_M60_G253_M60										88.3%	18.9
G253_M60_primary_unique					2.0%	86.8%	575 bp	1382 bp	42.6%
G253_M60_sorted			95.0%	17.9
G253_M60_statistics_for_all_accepted_reads	41.3	41.3
G253_M60_statistics_for_primary_reads	39.2	39.2
G253_M60_statistics_for_primary_unique_reads	37.7	37.7
G253_M61_G253_M61										88.8%	19.7
G253_M61_primary_unique					1.4%	84.3%	479 bp	1084 bp	39.4%
G253_M61_sorted			93.3%	18.4
G253_M61_statistics_for_all_accepted_reads	42.8	42.8
G253_M61_statistics_for_primary_reads	40.7	40.7
G253_M61_statistics_for_primary_unique_reads	39.3	39.3
G253_M62_G253_M62										88.4%	21.7
G253_M62_primary_unique					1.3%	87.9%	513 bp	1172 bp	41.4%
G253_M62_sorted			95.4%	20.7
G253_M62_statistics_for_all_accepted_reads	47.4	47.4
G253_M62_statistics_for_primary_reads	45.1	45.1
G253_M62_statistics_for_primary_unique_reads	43.5	43.5

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.

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:

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About MultiQC

General Statistics

General Statistics: Columns

RSeQC

Infer experiment

featureCounts

Picard

Insert Size

Mark Duplicates Help

RnaSeqMetrics Assignment

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