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 2024-07-19, 16:46 based on data in:

/projectnb/wax-dk/max/G227/SAMPLES
/projectnb/wax-dk/max/G227/Scripts/03_FASTQC
/projectnb/wax-dk/max/G227/Scripts/06_CollectMetrics

General Statistics

Showing ²⁴⁰/₂₄₀ rows and ¹⁴/₂₁ columns.

Sample Name	M Reads	M Reads Mapped	% Assigned	M Assigned	% rRNA	% mRNA	Insert Size	Mean Insert Size	% Aligned	M Aligned	% Duplication	% > Q30	Mb Q30 bases	GC content	% PF	% Adapter	% Dups	% GC	Length	% Failed	M Seqs
G227_M01_CKDL240023967-1A_22NF25LT3_L5_1											15.6%	94.5%	5036.0	53.1%	98.5%	73.8%	55.3%	54%	150 bp	27%	25.5
G227_M01_CKDL240023967-1A_22NF25LT3_L5_2																	54.1%	53%	150 bp	36%	25.5
G227_M01_G227_M01									60.8%	15.5
G227_M01_primary_unique					26.3%	16.1%	93 bp	113 bp
G227_M01_sorted			36.2%	5.6
G227_M01_statistics_for_all_accepted_reads	63.3	63.3
G227_M01_statistics_for_primary_reads	39.7	39.7
G227_M01_statistics_for_primary_unique_reads	29.8	29.8
G227_M02_CKDL240023967-1A_22NF25LT3_L5_1											36.4%	96.2%	4952.9	53.5%	98.8%	32.9%	62.6%	53%	150 bp	18%	18.7
G227_M02_CKDL240023967-1A_22NF25LT3_L5_2																	61.3%	53%	150 bp	36%	18.7
G227_M02_G227_M02									82.2%	15.4
G227_M02_primary_unique					39.4%	14.3%	172 bp	184 bp
G227_M02_sorted			59.9%	9.2
G227_M02_statistics_for_all_accepted_reads	40.4	40.4
G227_M02_statistics_for_primary_reads	33.8	33.8
G227_M02_statistics_for_primary_unique_reads	29.9	29.9
G227_M03_CKDL240023967-1A_22NF25LT3_L5_1											31.6%	95.7%	6342.2	55.0%	98.7%	59.4%	69.8%	55%	150 bp	18%	27.2
G227_M03_CKDL240023967-1A_22NF25LT3_L5_2																	69.0%	54%	150 bp	36%	27.2
G227_M03_G227_M03									71.9%	19.6
G227_M03_primary_unique					47.4%	14.6%	126 bp	144 bp
G227_M03_sorted			51.0%	10.0
G227_M03_statistics_for_all_accepted_reads	62.1	62.1
G227_M03_statistics_for_primary_reads	46.2	46.2
G227_M03_statistics_for_primary_unique_reads	37.8	37.8
G227_M04_CKDL240023967-1A_22NF25LT3_L5_1											18.2%	95.2%	5238.9	53.9%	98.3%	70.5%	57.8%	54%	150 bp	27%	25.7
G227_M04_CKDL240023967-1A_22NF25LT3_L5_2																	57.0%	54%	150 bp	36%	25.7
G227_M04_G227_M04									67.9%	17.5
G227_M04_primary_unique					35.4%	13.1%	101 bp	126 bp
G227_M04_sorted			47.8%	8.4
G227_M04_statistics_for_all_accepted_reads	64.3	64.3
G227_M04_statistics_for_primary_reads	43.2	43.2
G227_M04_statistics_for_primary_unique_reads	33.5	33.5
G227_M05_CKDL240023967-1A_22NF25LT3_L5_1											15.8%	95.7%	4947.7	52.1%	98.3%	78.0%	57.3%	54%	150 bp	36%	24.7
G227_M05_CKDL240023967-1A_22NF25LT3_L5_2																	56.4%	52%	150 bp	27%	24.7
G227_M05_G227_M05									62.8%	15.5
G227_M05_primary_unique					31.3%	12.8%	92 bp	108 bp
G227_M05_sorted			42.3%	6.6
G227_M05_statistics_for_all_accepted_reads	56.9	56.9
G227_M05_statistics_for_primary_reads	38.3	38.3
G227_M05_statistics_for_primary_unique_reads	29.7	29.7
G227_M06_CKDL240023967-1A_22NF25LT3_L5_1											29.6%	95.4%	5863.6	58.6%	98.5%	68.3%	71.6%	57%	150 bp	27%	26.9
G227_M06_CKDL240023967-1A_22NF25LT3_L5_2																	71.0%	57%	150 bp	45%	26.9
G227_M06_G227_M06									72.0%	19.4
G227_M06_primary_unique					46.2%	19.0%	104 bp	118 bp
G227_M06_sorted			45.8%	8.9
G227_M06_statistics_for_all_accepted_reads	64.8	64.8
G227_M06_statistics_for_primary_reads	46.4	46.4
G227_M06_statistics_for_primary_unique_reads	37.3	37.3
G227_M07_CKDL240023967-1A_22NF25LT3_L5_1											25.6%	95.9%	5422.9	51.0%	98.7%	58.7%	59.6%	51%	150 bp	27%	23.1
G227_M07_CKDL240023967-1A_22NF25LT3_L5_2																	58.7%	51%	150 bp	36%	23.1
G227_M07_G227_M07									66.6%	15.4
G227_M07_primary_unique					35.9%	12.0%	129 bp	148 bp
G227_M07_sorted			49.5%	7.6
G227_M07_statistics_for_all_accepted_reads	50.0	50.0
G227_M07_statistics_for_primary_reads	36.4	36.4
G227_M07_statistics_for_primary_unique_reads	29.6	29.6
G227_M08_CKDL240023967-1A_22NF25LT3_L5_1											29.5%	96.0%	5132.3	51.0%	98.7%	53.9%	67.5%	51%	150 bp	27%	21.3
G227_M08_CKDL240023967-1A_22NF25LT3_L5_2																	66.1%	51%	150 bp	36%	21.3
G227_M08_G227_M08									64.4%	13.7
G227_M08_primary_unique					41.0%	12.9%	145 bp	162 bp
G227_M08_sorted			50.5%	6.9
G227_M08_statistics_for_all_accepted_reads	44.7	44.7
G227_M08_statistics_for_primary_reads	32.5	32.5
G227_M08_statistics_for_primary_unique_reads	26.6	26.6
G227_M09_CKDL240023967-1A_22NF25LT3_L5_1											40.6%	95.4%	5345.6	59.5%	98.4%	35.0%	74.0%	58%	150 bp	27%	20.6
G227_M09_CKDL240023967-1A_22NF25LT3_L5_2																	73.3%	59%	150 bp	27%	20.6
G227_M09_G227_M09									81.8%	16.9
G227_M09_primary_unique					45.5%	20.6%	152 bp	158 bp
G227_M09_sorted			45.5%	7.7
G227_M09_statistics_for_all_accepted_reads	44.8	44.8
G227_M09_statistics_for_primary_reads	37.1	37.1
G227_M09_statistics_for_primary_unique_reads	32.4	32.4
G227_M10_CKDL240023967-1A_22NF25LT3_L5_1											27.2%	95.2%	4907.7	55.8%	98.2%	58.1%	62.0%	56%	150 bp	18%	21.1
G227_M10_CKDL240023967-1A_22NF25LT3_L5_2																	61.5%	55%	150 bp	18%	21.1
G227_M10_G227_M10									73.9%	15.6
G227_M10_primary_unique					39.2%	15.9%	121 bp	138 bp
G227_M10_sorted			44.4%	6.9
G227_M10_statistics_for_all_accepted_reads	47.5	47.5
G227_M10_statistics_for_primary_reads	36.0	36.0
G227_M10_statistics_for_primary_unique_reads	30.0	30.0
G227_M11_CKDL240023967-1A_22NF25LT3_L5_1											27.7%	93.1%	4159.0	59.5%	98.4%	53.6%	62.5%	58%	150 bp	45%	18.5
G227_M11_CKDL240023967-1A_22NF25LT3_L5_2																	61.6%	59%	150 bp	45%	18.5
G227_M11_G227_M11									67.9%	12.6
G227_M11_primary_unique					32.9%	17.8%	125 bp	143 bp
G227_M11_sorted			51.3%	6.4
G227_M11_statistics_for_all_accepted_reads	48.4	48.4
G227_M11_statistics_for_primary_reads	31.5	31.5
G227_M11_statistics_for_primary_unique_reads	24.1	24.1
G227_M12_CKDL240023967-1A_22NF25LT3_L5_1											36.3%	96.1%	5175.2	57.6%	98.8%	52.8%	68.4%	57%	150 bp	27%	21.0
G227_M12_CKDL240023967-1A_22NF25LT3_L5_2																	67.8%	57%	150 bp	36%	21.0
G227_M12_G227_M12									80.8%	17.0
G227_M12_primary_unique					41.1%	18.3%	133 bp	144 bp
G227_M12_sorted			54.8%	9.3
G227_M12_statistics_for_all_accepted_reads	47.7	47.7
G227_M12_statistics_for_primary_reads	38.5	38.5
G227_M12_statistics_for_primary_unique_reads	32.9	32.9
G227_M13_CKDL240023967-1A_22NF25LT3_L5_1											17.4%	95.5%	5155.5	52.8%	98.5%	70.4%	48.0%	54%	150 bp	18%	24.7
G227_M13_CKDL240023967-1A_22NF25LT3_L5_2																	47.5%	53%	150 bp	18%	24.7
G227_M13_G227_M13									72.2%	17.8
G227_M13_primary_unique					22.8%	15.7%	105 bp	127 bp
G227_M13_sorted			46.7%	8.3
G227_M13_statistics_for_all_accepted_reads	58.7	58.7
G227_M13_statistics_for_primary_reads	42.2	42.2
G227_M13_statistics_for_primary_unique_reads	34.3	34.3
G227_M14_CKDL240023967-1A_22NF25LT3_L5_1											26.4%	95.3%	4866.6	54.9%	98.1%	54.1%	55.2%	54%	150 bp	18%	20.0
G227_M14_CKDL240023967-1A_22NF25LT3_L5_2																	55.0%	54%	150 bp	36%	20.0
G227_M14_G227_M14									76.8%	15.3
G227_M14_primary_unique					30.8%	14.0%	132 bp	146 bp
G227_M14_sorted			44.7%	6.8
G227_M14_statistics_for_all_accepted_reads	43.9	43.9
G227_M14_statistics_for_primary_reads	34.7	34.7
G227_M14_statistics_for_primary_unique_reads	29.4	29.4
G227_M15_CKDL240023967-1A_22NF25LT3_L5_1											18.2%	95.8%	6183.8	51.1%	98.4%	65.1%	50.1%	51%	150 bp	18%	27.7
G227_M15_CKDL240023967-1A_22NF25LT3_L5_2																	48.6%	51%	150 bp	9%	27.7
G227_M15_G227_M15									71.2%	19.7
G227_M15_primary_unique					25.2%	13.7%	121 bp	142 bp
G227_M15_sorted			49.7%	9.8
G227_M15_statistics_for_all_accepted_reads	61.5	61.5
G227_M15_statistics_for_primary_reads	45.8	45.8
G227_M15_statistics_for_primary_unique_reads	37.8	37.8
G227_M16_CKDL240023967-1A_22NF25LT3_L5_1											35.5%	95.5%	4857.7	57.6%	98.4%	47.8%	64.8%	57%	150 bp	27%	19.4
G227_M16_CKDL240023967-1A_22NF25LT3_L5_2																	65.1%	57%	150 bp	36%	19.4
G227_M16_G227_M16									81.8%	15.9
G227_M16_primary_unique					34.6%	22.4%	138 bp	148 bp
G227_M16_sorted			51.4%	8.2
G227_M16_statistics_for_all_accepted_reads	43.0	43.0
G227_M16_statistics_for_primary_reads	35.3	35.3
G227_M16_statistics_for_primary_unique_reads	30.6	30.6
G227_M17_CKDL240023967-1A_22NF25LT3_L5_1											20.2%	94.8%	6255.0	52.4%	98.3%	63.9%	50.7%	53%	150 bp	18%	27.8
G227_M17_CKDL240023967-1A_22NF25LT3_L5_2																	50.3%	53%	150 bp	45%	27.8
G227_M17_G227_M17									69.0%	19.2
G227_M17_primary_unique					22.3%	15.1%	119 bp	135 bp
G227_M17_sorted			42.5%	8.1
G227_M17_statistics_for_all_accepted_reads	63.8	63.8
G227_M17_statistics_for_primary_reads	45.5	45.5
G227_M17_statistics_for_primary_unique_reads	36.9	36.9
G227_M18_CKDL240023967-1A_22NF25LT3_L5_1											24.6%	95.2%	5819.2	50.3%	98.2%	49.6%	51.5%	50%	150 bp	18%	23.9
G227_M18_CKDL240023967-1A_22NF25LT3_L5_2																	51.5%	50%	150 bp	36%	23.9
G227_M18_G227_M18									71.0%	17.0
G227_M18_primary_unique					27.6%	11.0%	143 bp	159 bp
G227_M18_sorted			42.1%	7.2
G227_M18_statistics_for_all_accepted_reads	50.3	50.3
G227_M18_statistics_for_primary_reads	38.8	38.8
G227_M18_statistics_for_primary_unique_reads	32.8	32.8
G227_M19_CKDL240023967-1A_22NF25LT3_L5_1											23.1%	95.7%	6465.0	54.3%	98.4%	64.7%	56.4%	54%	150 bp	27%	29.2
G227_M19_CKDL240023967-1A_22NF25LT3_L5_2																	55.5%	54%	150 bp	27%	29.2
G227_M19_G227_M19									73.9%	21.6
G227_M19_primary_unique					31.1%	15.8%	115 bp	134 bp
G227_M19_sorted			47.1%	10.2
G227_M19_statistics_for_all_accepted_reads	66.5	66.5
G227_M19_statistics_for_primary_reads	50.2	50.2
G227_M19_statistics_for_primary_unique_reads	41.5	41.5
G227_M20_CKDL240023967-1A_22NF25LT3_L5_1											16.4%	95.6%	5733.9	49.3%	98.4%	56.4%	41.4%	50%	150 bp	9%	24.7
G227_M20_CKDL240023967-1A_22NF25LT3_L5_2																	40.4%	50%	150 bp	9%	24.7
G227_M20_G227_M20									71.7%	17.7
G227_M20_primary_unique					17.0%	9.7%	140 bp	157 bp
G227_M20_sorted			45.8%	8.1
G227_M20_statistics_for_all_accepted_reads	53.6	53.6
G227_M20_statistics_for_primary_reads	40.5	40.5
G227_M20_statistics_for_primary_unique_reads	34.0	34.0
G227_M21_CKDL240023967-1A_22NF25LT3_L5_1											20.0%	94.7%	5938.0	50.9%	98.4%	63.5%	50.9%	52%	150 bp	36%	26.2
G227_M21_CKDL240023967-1A_22NF25LT3_L5_2																	49.9%	51%	150 bp	18%	26.2
G227_M21_G227_M21									66.9%	17.5
G227_M21_primary_unique					24.7%	12.6%	125 bp	144 bp
G227_M21_sorted			44.2%	7.7
G227_M21_statistics_for_all_accepted_reads	60.1	60.1
G227_M21_statistics_for_primary_reads	42.0	42.0
G227_M21_statistics_for_primary_unique_reads	33.7	33.7
G227_M22_CKDL240023967-1A_22NF25LT3_L5_1											18.1%	95.0%	5484.2	50.8%	98.5%	60.7%	46.8%	52%	150 bp	18%	24.6
G227_M22_CKDL240023967-1A_22NF25LT3_L5_2																	45.5%	51%	150 bp	27%	24.6
G227_M22_G227_M22									70.6%	17.4
G227_M22_primary_unique					21.3%	13.3%	131 bp	152 bp
G227_M22_sorted			52.3%	9.1
G227_M22_statistics_for_all_accepted_reads	58.0	58.0
G227_M22_statistics_for_primary_reads	41.3	41.3
G227_M22_statistics_for_primary_unique_reads	33.6	33.6
G227_M23_CKDL240023967-1A_22NF25LT3_L5_1											19.3%	94.1%	5619.9	55.1%	98.3%	72.7%	57.9%	55%	150 bp	27%	28.3
G227_M23_CKDL240023967-1A_22NF25LT3_L5_2																	57.4%	55%	150 bp	36%	28.3
G227_M23_G227_M23									65.0%	18.4
G227_M23_primary_unique					33.6%	14.8%	95 bp	112 bp
G227_M23_sorted			37.1%	6.8
G227_M23_statistics_for_all_accepted_reads	71.9	71.9
G227_M23_statistics_for_primary_reads	46.6	46.6
G227_M23_statistics_for_primary_unique_reads	35.3	35.3
G227_M24_CKDL240023967-1A_22NF25LT3_L5_1											6.0%	94.6%	5115.1	47.2%	98.4%	82.8%	41.3%	53%	150 bp	27%	29.8
G227_M24_CKDL240023967-1A_22NF25LT3_L5_2																	40.1%	50%	150 bp	36%	29.8
G227_M24_G227_M24									56.2%	16.8
G227_M24_primary_unique					8.2%	5.8%	79 bp	102 bp
G227_M24_sorted			37.0%	6.2
G227_M24_statistics_for_all_accepted_reads	73.5	73.5
G227_M24_statistics_for_primary_reads	43.9	43.9
G227_M24_statistics_for_primary_unique_reads	32.0	32.0
G227_M25_CKDL240023967-1A_22NF25LT3_L5_1											10.9%	94.6%	6148.0	43.6%	98.0%	50.4%	41.2%	44%	150 bp	27%	26.2
G227_M25_CKDL240023967-1A_22NF25LT3_L5_2																	41.0%	45%	150 bp	27%	26.2
G227_M25_G227_M25									58.5%	15.4
G227_M25_primary_unique					4.3%	3.8%	149 bp	176 bp
G227_M25_sorted			34.7%	5.3
G227_M25_statistics_for_all_accepted_reads	50.6	50.6
G227_M25_statistics_for_primary_reads	35.9	35.9
G227_M25_statistics_for_primary_unique_reads	29.5	29.5
G227_M26_CKDL240023967-1A_22NF25LT3_L5_1											10.7%	93.9%	4441.1	48.4%	97.5%	73.6%	48.0%	51%	150 bp	27%	22.3
G227_M26_CKDL240023967-1A_22NF25LT3_L5_2																	49.6%	49%	150 bp	27%	22.3
G227_M26_G227_M26									60.5%	13.5
G227_M26_primary_unique					20.2%	7.1%	96 bp	118 bp
G227_M26_sorted			32.7%	4.4
G227_M26_statistics_for_all_accepted_reads	51.0	51.0
G227_M26_statistics_for_primary_reads	33.4	33.4
G227_M26_statistics_for_primary_unique_reads	25.8	25.8
G227_M27_CKDL240023967-1A_22NF25LT3_L5_1											18.0%	94.9%	5591.5	50.8%	98.4%	61.8%	44.7%	52%	150 bp	18%	25.0
G227_M27_CKDL240023967-1A_22NF25LT3_L5_2																	43.9%	51%	150 bp	18%	25.0
G227_M27_G227_M27									71.2%	17.8
G227_M27_primary_unique					22.5%	11.4%	127 bp	148 bp
G227_M27_sorted			44.9%	8.0
G227_M27_statistics_for_all_accepted_reads	58.9	58.9
G227_M27_statistics_for_primary_reads	42.1	42.1
G227_M27_statistics_for_primary_unique_reads	34.2	34.2
G227_M28_CKDL240023967-1A_22NF25LT3_L5_1											6.5%	94.5%	4929.2	48.2%	98.0%	79.0%	36.5%	52%	150 bp	18%	27.5
G227_M28_CKDL240023967-1A_22NF25LT3_L5_2																	36.7%	50%	150 bp	18%	27.5
G227_M28_G227_M28									64.0%	17.6
G227_M28_primary_unique					9.1%	6.8%	86 bp	113 bp
G227_M28_sorted			40.2%	7.1
G227_M28_statistics_for_all_accepted_reads	67.9	67.9
G227_M28_statistics_for_primary_reads	43.9	43.9
G227_M28_statistics_for_primary_unique_reads	33.7	33.7
G227_M29_CKDL240023967-1A_22NF25LT3_L5_1											16.8%	95.8%	5896.9	47.3%	98.4%	51.5%	39.0%	48%	150 bp	9%	24.3
G227_M29_CKDL240023967-1A_22NF25LT3_L5_2																	37.9%	48%	150 bp	18%	24.3
G227_M29_G227_M29									73.6%	17.9
G227_M29_primary_unique					13.2%	10.4%	153 bp	169 bp
G227_M29_sorted			53.4%	9.5
G227_M29_statistics_for_all_accepted_reads	52.5	52.5
G227_M29_statistics_for_primary_reads	40.4	40.4
G227_M29_statistics_for_primary_unique_reads	34.6	34.6
G227_M30_CKDL240023967-1A_22NF25LT3_L5_1											4.8%	94.7%	4691.5	44.3%	98.2%	87.5%	47.2%	52%	150 bp	27%	28.1
G227_M30_CKDL240023967-1A_22NF25LT3_L5_2																	46.0%	48%	150 bp	27%	28.1
G227_M30_G227_M30									48.0%	13.5
G227_M30_primary_unique					2.4%	2.1%	76 bp	94 bp
G227_M30_sorted			30.0%	4.1
G227_M30_statistics_for_all_accepted_reads	67.2	67.2
G227_M30_statistics_for_primary_reads	37.1	37.1
G227_M30_statistics_for_primary_unique_reads	25.9	25.9

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
\|\|	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.

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

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 Counts

Sequence counts for each sample. Duplicate read counts are an estimate only.

This plot show the total number of reads, broken down into unique and duplicate if possible (only more recent versions of FastQC give duplicate info).

You can read more about duplicate calculation in the FastQC documentation. A small part has been copied here for convenience:

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.

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.

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.

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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General Statistics

General Statistics: Columns

RSeQC

Infer experiment

featureCounts

Picard

Insert Size

RnaSeqMetrics Assignment

RnaSeqMetrics Strand Mapping

Gene Coverage

Samtools

Samtools Flagstat

STAR

Alignment Scores

fastp

Filtered Reads

Insert Sizes

Sequence Quality

GC Content

N content

FastQC

Sequence Counts Help

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

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Sequence Counts

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Status Checks