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 2024-12-13, 15:33 based on data in: /scratch/1543031.1.linga/nxf.7ALYDphfvA

General Statistics

Showing ²⁸⁰/₂₈₀ rows and ¹⁶/₂₀ columns.

Sample Name	M Reads	M Reads Mapped	Fragment Length	Number of Peaks	% Aligned	Insert Size	Mean Insert Size	Error rate	M Non-Primary	M Reads Mapped	% Mapped	% Proper Pairs	% MapQ 0 Reads	M Total seqs	% Aligned	% Dups	% GC	Read Length	% Failed	M Seqs
G222_M09															95.5%
G222_M09_1																13.0%	51%	146 bp	0%	15.0
G222_M09_2																12.1%	51%	146 bp	0%	15.0
G222_M09_narrow_MACS2			256	41174
G222_M09_sorted_filtered	23.0	23.0			100%	244 bp	257 bp	0.77%	0.0	23.0	100.0%	100.0%	0.2%	23.0
G222_M10															94.9%
G222_M10_1																14.2%	50%	145 bp	0%	10.2
G222_M10_2																13.5%	50%	145 bp	0%	10.2
G222_M10_narrow_MACS2			237	29329
G222_M10_sorted_filtered	14.7	14.7			100%	226 bp	237 bp	0.79%	0.0	14.7	100.0%	100.0%	0.3%	14.7
G222_M11															96.1%
G222_M11_1																15.0%	50%	146 bp	0%	13.9
G222_M11_2																14.2%	51%	146 bp	0%	13.9
G222_M11_narrow_MACS2			245	37763
G222_M11_sorted_filtered	20.7	20.7			100%	235 bp	246 bp	0.79%	0.0	20.7	100.0%	100.0%	0.2%	20.7
G222_M12															95.9%
G222_M12_1																15.9%	48%	143 bp	0%	16.3
G222_M12_2																15.3%	48%	143 bp	0%	16.3
G222_M12_narrow_MACS2			226	35465
G222_M12_sorted_filtered	23.3	23.3			100%	216 bp	226 bp	0.74%	0.0	23.3	100.0%	100.0%	0.3%	23.3
G222_M16															96.9%
G222_M16_1																13.2%	48%	147 bp	0%	19.1
G222_M16_2																11.4%	48%	147 bp	0%	19.1
G222_M16_narrow_MACS2			283	43570
G222_M16_sorted_filtered	29.4	29.4			100%	273 bp	283 bp	0.81%	0.0	29.4	100.0%	100.0%	0.1%	29.4
G222_M17															96.8%
G222_M17_1																15.9%	43%	147 bp	9%	21.8
G222_M17_2																14.1%	44%	147 bp	9%	21.8
G222_M17_narrow_MACS2			289	27585
G222_M17_sorted_filtered	30.1	30.1			100%	280 bp	290 bp	0.82%	0.0	30.1	100.0%	100.0%	0.2%	30.1
G222_M18															96.5%
G222_M18_1																15.4%	44%	147 bp	0%	10.9
G222_M18_2																13.5%	44%	147 bp	0%	10.9
G222_M18_narrow_MACS2			330	21219
G222_M18_sorted_filtered	14.2	14.2			100%	329 bp	330 bp	0.85%	0.0	14.2	100.0%	100.0%	0.1%	14.2
G222_M19															97.1%
G222_M19_1																15.0%	46%	147 bp	0%	20.3
G222_M19_2																13.8%	46%	147 bp	0%	20.3
G222_M19_narrow_MACS2			297	40779
G222_M19_sorted_filtered	29.9	29.9			100%	291 bp	298 bp	0.77%	0.0	29.9	100.0%	100.0%	0.1%	29.9
G222_M20															96.8%
G222_M20_1																17.4%	45%	146 bp	0%	15.5
G222_M20_2																16.1%	45%	146 bp	0%	15.5
G222_M20_narrow_MACS2			297	27932
G222_M20_sorted_filtered	20.6	20.6			100%	292 bp	298 bp	0.81%	0.0	20.6	100.0%	100.0%	0.2%	20.6
G223_M01D															91.7%
G223_M01D_1																17.6%	47%	145 bp	0%	58.0
G223_M01D_2																15.9%	48%	145 bp	9%	58.0
G223_M01D_narrow_MACS2			262	66505
G223_M01D_sorted_filtered	75.5	75.5			100%	253 bp	262 bp	1.05%	0.0	75.5	100.0%	100.0%	0.7%	75.5
G223_M02															85.6%
G223_M02_1																9.4%	48%	140 bp	0%	13.1
G223_M02_2																9.4%	49%	140 bp	9%	13.1
G223_M02_narrow_MACS2			247	34579
G223_M02_sorted_filtered	15.1	15.1			100%	236 bp	248 bp	2.39%	0.0	15.1	100.0%	100.0%	2.5%	15.1
G223_M03															84.0%
G223_M03_1																12.2%	48%	142 bp	0%	26.1
G223_M03_2																11.5%	49%	142 bp	9%	26.1
G223_M03_narrow_MACS2			243	46513
G223_M03_sorted_filtered	29.8	29.8			100%	232 bp	244 bp	2.77%	0.0	29.8	100.0%	100.0%	2.4%	29.8
G223_M04															83.2%
G223_M04_1																14.4%	47%	142 bp	0%	53.9
G223_M04_2																11.8%	48%	142 bp	18%	53.9
G223_M04_narrow_MACS2			241	61842
G223_M04_sorted_filtered	54.2	54.2			100%	231 bp	241 bp	2.14%	0.0	54.2	100.0%	100.0%	2.2%	54.2
G223_M05															84.9%
G223_M05_1																10.6%	47%	144 bp	0%	9.4
G223_M05_2																10.7%	48%	144 bp	9%	9.4
G223_M05_narrow_MACS2			257	28006
G223_M05_sorted_filtered	10.6	10.6			100%	246 bp	257 bp	2.10%	0.0	10.6	100.0%	100.0%	1.6%	10.6
G223_M06															81.9%
G223_M06_1																12.3%	48%	141 bp	9%	24.1
G223_M06_2																12.0%	50%	141 bp	18%	24.1
G223_M06_narrow_MACS2			241	43688
G223_M06_sorted_filtered	25.1	25.1			100%	226 bp	241 bp	2.73%	0.0	25.1	100.0%	100.0%	2.9%	25.1
G223_M07															85.5%
G223_M07_1																12.0%	52%	144 bp	0%	11.1
G223_M07_2																11.4%	52%	144 bp	9%	11.1
G223_M07_narrow_MACS2			233	30735
G223_M07_sorted_filtered	13.4	13.4			100%	223 bp	233 bp	2.49%	0.0	13.4	100.0%	100.0%	2.0%	13.4
G223_M08															88.3%
G223_M08_1																16.9%	53%	144 bp	9%	31.0
G223_M08_2																16.3%	54%	144 bp	9%	31.0
G223_M08_narrow_MACS2			259	59377
G223_M08_sorted_filtered	40.5	40.5			100%	249 bp	260 bp	1.52%	0.0	40.5	100.0%	100.0%	0.9%	40.5
G223_M09															90.2%
G223_M09_1																17.6%	52%	143 bp	9%	27.9
G223_M09_2																16.6%	53%	142 bp	9%	27.9
G223_M09_narrow_MACS2			257	56484
G223_M09_sorted_filtered	36.6	36.6			100%	247 bp	258 bp	1.09%	0.0	36.6	100.0%	100.0%	0.9%	36.6
G223_M10															89.7%
G223_M10_1																16.3%	48%	145 bp	0%	32.3
G223_M10_2																15.0%	48%	145 bp	9%	32.3
G223_M10_narrow_MACS2			257	54508
G223_M10_sorted_filtered	38.8	38.8			100%	247 bp	257 bp	1.30%	0.0	38.8	100.0%	100.0%	1.0%	38.8
G223_M11															82.8%
G223_M11_1																18.8%	47%	138 bp	9%	33.8
G223_M11_2																17.2%	48%	138 bp	27%	33.8
G223_M11_narrow_MACS2			230	50150
G223_M11_sorted_filtered	34.3	34.3			100%	216 bp	230 bp	1.45%	0.0	34.3	100.0%	100.0%	2.0%	34.3
G223_M12															87.8%
G223_M12_1																19.3%	47%	143 bp	9%	41.0
G223_M12_2																17.3%	47%	142 bp	18%	41.0
G223_M12_narrow_MACS2			250	53654
G223_M12_sorted_filtered	45.5	45.5			100%	240 bp	251 bp	1.40%	0.0	45.5	100.0%	100.0%	1.5%	45.5
G223_M13M14															84.9%
G223_M13M14_1																21.6%	48%	142 bp	9%	35.9
G223_M13M14_2																19.9%	49%	142 bp	18%	35.9
G223_M13M14_narrow_MACS2			226	49011
G223_M13M14_sorted_filtered	38.8	38.8			100%	215 bp	226 bp	1.98%	0.0	38.8	100.0%	100.0%	2.0%	38.8
G223_M16															82.1%
G223_M16_1																10.6%	47%	142 bp	0%	9.7
G223_M16_2																11.0%	48%	142 bp	18%	9.7
G223_M16_narrow_MACS2			242	26111
G223_M16_sorted_filtered	10.0	10.0			100%	229 bp	242 bp	2.51%	0.0	10.0	100.0%	100.0%	2.1%	10.0
G223_M17															80.8%
G223_M17_1																10.8%	46%	142 bp	0%	9.7
G223_M17_2																10.8%	47%	142 bp	9%	9.7
G223_M17_narrow_MACS2			242	27051
G223_M17_sorted_filtered	10.0	10.0			100%	232 bp	243 bp	2.84%	0.0	10.0	100.0%	100.0%	2.0%	10.0
G223_M18															77.6%
G223_M18_1																29.8%	47%	141 bp	9%	8.9
G223_M18_2																30.6%	49%	141 bp	36%	8.9
G223_M18_narrow_MACS2			217	47332
G223_M18_sorted_filtered	9.6	9.6			100%	210 bp	218 bp	3.36%	0.0	9.6	100.0%	100.0%	3.1%	9.6
G223_M19															82.6%
G223_M19_1																10.4%	48%	143 bp	0%	9.2
G223_M19_2																10.2%	48%	143 bp	18%	9.2
G223_M19_narrow_MACS2			264	25199
G223_M19_sorted_filtered	10.0	10.0			100%	255 bp	264 bp	2.19%	0.0	10.0	100.0%	100.0%	1.7%	10.0
G223_M20															85.4%
G223_M20_1																15.7%	52%	141 bp	0%	23.3
G223_M20_2																14.6%	52%	141 bp	9%	23.3
G223_M20_narrow_MACS2			233	44997
G223_M20_sorted_filtered	27.7	27.7			100%	220 bp	233 bp	2.10%	0.0	27.7	100.0%	100.0%	2.2%	27.7
G223_M21															92.0%
G223_M21_1																14.5%	48%	146 bp	0%	16.5
G223_M21_2																14.3%	48%	146 bp	27%	16.5
G223_M21_narrow_MACS2			300	37007
G223_M21_sorted_filtered	21.1	21.1			100%	293 bp	300 bp	1.07%	0.0	21.1	100.0%	100.0%	0.4%	21.1
G223_M22															87.0%
G223_M22_1																13.6%	48%	144 bp	0%	23.8
G223_M22_2																13.3%	49%	144 bp	18%	23.8
G223_M22_narrow_MACS2			281	40612
G223_M22_sorted_filtered	27.6	27.6			100%	273 bp	281 bp	1.99%	0.0	27.6	100.0%	100.0%	1.4%	27.6
G223_M23															84.9%
G223_M23_1																9.7%	49%	145 bp	0%	12.7
G223_M23_2																9.6%	50%	145 bp	9%	12.7
G223_M23_narrow_MACS2			252	38275
G223_M23_sorted_filtered	16.0	16.0			100%	242 bp	253 bp	2.43%	0.0	16.0	100.0%	100.0%	1.3%	16.0
G223_M24															82.5%
G223_M24_1																12.3%	47%	140 bp	0%	39.0
G223_M24_2																11.3%	48%	139 bp	9%	39.0
G223_M24_narrow_MACS2			243	56659
G223_M24_sorted_filtered	42.5	42.5			100%	228 bp	243 bp	2.82%	0.0	42.5	100.0%	100.0%	3.4%	42.5
G223_M25D															90.0%
G223_M25D_1																19.3%	52%	143 bp	0%	35.1
G223_M25D_2																17.4%	52%	143 bp	9%	35.1
G223_M25D_narrow_MACS2			247	47444
G223_M25D_sorted_filtered	44.2	44.2			100%	233 bp	247 bp	1.37%	0.0	44.2	100.0%	100.0%	1.0%	44.2
G223_M26															90.9%
G223_M26_1																16.3%	53%	146 bp	0%	15.7
G223_M26_2																16.1%	53%	146 bp	18%	15.7
G223_M26_narrow_MACS2			275	35923
G223_M26_sorted_filtered	20.3	20.3			100%	264 bp	275 bp	1.26%	0.0	20.3	100.0%	100.0%	0.6%	20.3
G223_M27															86.5%
G223_M27_1																14.9%	47%	145 bp	0%	45.8
G223_M27_2																14.6%	49%	145 bp	18%	45.8
G223_M27_narrow_MACS2			261	55926
G223_M27_sorted_filtered	53.7	53.7			100%	247 bp	262 bp	3.03%	0.0	53.7	100.0%	100.0%	1.5%	53.7
G223_M28															84.6%
G223_M28_1																8.5%	47%	143 bp	9%	2.9
G223_M28_2																9.1%	48%	143 bp	18%	2.9
G223_M28_narrow_MACS2			264	15834
G223_M28_sorted_filtered	3.4	3.4			100%	252 bp	264 bp	3.06%	0.0	3.4	100.0%	100.0%	2.2%	3.4
G223_M29															87.9%
G223_M29_1																14.4%	48%	144 bp	0%	18.5
G223_M29_2																14.0%	48%	143 bp	18%	18.5
G223_M29_narrow_MACS2			279	37563
G223_M29_sorted_filtered	20.9	20.9			100%	270 bp	279 bp	1.86%	0.0	20.9	100.0%	100.0%	1.1%	20.9
G223_M30D															88.6%
G223_M30D_1																15.4%	47%	145 bp	0%	38.7
G223_M30D_2																14.2%	48%	145 bp	18%	38.7
G223_M30D_narrow_MACS2			256	49418
G223_M30D_sorted_filtered	45.0	45.0			100%	245 bp	257 bp	1.47%	0.0	45.0	100.0%	100.0%	0.9%	45.0
G223_M31															79.7%
G223_M31_1																12.7%	48%	139 bp	9%	13.3
G223_M31_2																11.1%	48%	138 bp	9%	13.3
G223_M31_narrow_MACS2			216	32985
G223_M31_sorted_filtered	14.1	14.1			100%	205 bp	216 bp	3.48%	0.0	14.1	100.0%	100.0%	3.6%	14.1
G223_M32															84.1%
G223_M32_1																10.4%	46%	143 bp	0%	9.3
G223_M32_2																9.8%	47%	143 bp	9%	9.3
G223_M32_narrow_MACS2			245	29585
G223_M32_sorted_filtered	10.4	10.4			100%	230 bp	245 bp	2.93%	0.0	10.4	100.0%	100.0%	2.4%	10.4
G223_M33															82.1%
G223_M33_1																9.8%	46%	142 bp	0%	7.4
G223_M33_2																8.9%	47%	141 bp	9%	7.4
G223_M33_narrow_MACS2			226	28319
G223_M33_sorted_filtered	8.5	8.5			100%	215 bp	226 bp	3.62%	0.0	8.5	100.0%	100.0%	2.9%	8.5
G223_M34															86.6%
G223_M34_1																14.4%	50%	141 bp	9%	13.8
G223_M34_2																13.2%	51%	141 bp	18%	13.8
G223_M34_narrow_MACS2			226	30184
G223_M34_sorted_filtered	16.3	16.3			100%	217 bp	227 bp	1.99%	0.0	16.3	100.0%	100.0%	2.2%	16.3
G223_M35															79.3%
G223_M35_1																10.3%	49%	142 bp	9%	4.4
G223_M35_2																9.4%	49%	141 bp	18%	4.4
G223_M35_narrow_MACS2			239	15819
G223_M35_sorted_filtered	4.6	4.6			100%	230 bp	240 bp	2.98%	0.0	4.6	100.0%	100.0%	2.7%	4.6
G223_M36															85.6%
G223_M36_1																14.9%	48%	140 bp	0%	22.5
G223_M36_2																13.1%	48%	140 bp	9%	22.5
G223_M36_narrow_MACS2			221	47369
G223_M36_sorted_filtered	26.2	26.2			100%	210 bp	222 bp	1.62%	0.0	26.2	100.0%	100.0%	2.0%	26.2
G223_M37															80.9%
G223_M37_1																10.1%	47%	139 bp	0%	12.1
G223_M37_2																9.3%	48%	139 bp	9%	12.1
G223_M37_narrow_MACS2			220	33442
G223_M37_sorted_filtered	12.7	12.7			100%	208 bp	221 bp	2.85%	0.0	12.7	100.0%	100.0%	3.3%	12.7
G223_M38															84.3%
G223_M38_1																15.7%	46%	142 bp	0%	28.7
G223_M38_2																13.8%	47%	141 bp	9%	28.7
G223_M38_narrow_MACS2			238	46220
G223_M38_sorted_filtered	30.6	30.6			100%	231 bp	239 bp	2.04%	0.0	30.6	100.0%	100.0%	1.7%	30.6
G223_M39															81.2%
G223_M39_1																10.1%	46%	145 bp	9%	4.4
G223_M39_2																10.0%	48%	145 bp	18%	4.4
G223_M39_narrow_MACS2			245	16765
G223_M39_sorted_filtered	5.0	5.0			100%	234 bp	245 bp	3.26%	0.0	5.0	100.0%	100.0%	1.7%	5.0
G223_M40															83.2%
G223_M40_1																11.2%	49%	145 bp	9%	5.7
G223_M40_2																11.6%	50%	145 bp	18%	5.7
G223_M40_narrow_MACS2			251	21701
G223_M40_sorted_filtered	6.4	6.4			100%	240 bp	251 bp	2.71%	0.0	6.4	100.0%	100.0%	1.5%	6.4
G223_M41															84.2%
G223_M41_1																8.9%	46%	142 bp	0%	10.8
G223_M41_2																8.1%	47%	142 bp	9%	10.8
G223_M41_narrow_MACS2			222	35660
G223_M41_sorted_filtered	13.0	13.0			100%	214 bp	222 bp	3.85%	0.0	13.0	100.0%	100.0%	2.7%	13.0
G223_M42															91.6%
G223_M42_1																16.2%	52%	144 bp	0%	26.9
G223_M42_2																15.1%	52%	144 bp	9%	26.9
G223_M42_narrow_MACS2			247	57359
G223_M42_sorted_filtered	37.0	37.0			100%	237 bp	247 bp	0.92%	0.0	37.0	100.0%	100.0%	0.7%	37.0
G223_M43D															90.1%
G223_M43D_1																18.5%	51%	145 bp	0%	39.2
G223_M43D_2																17.3%	52%	145 bp	9%	39.2
G223_M43D_narrow_MACS2			255	61106
G223_M43D_sorted_filtered	49.7	49.7			100%	245 bp	256 bp	1.05%	0.0	49.7	100.0%	100.0%	0.7%	49.7
G223_M44															83.2%
G223_M44_1																10.7%	52%	145 bp	9%	6.0
G223_M44_2																11.1%	53%	145 bp	18%	6.0
G223_M44_narrow_MACS2			249	21866
G223_M44_sorted_filtered	6.9	6.9			100%	237 bp	249 bp	2.13%	0.0	6.9	100.0%	100.0%	1.2%	6.9
G223_M45															86.1%
G223_M45_1																25.4%	50%	145 bp	9%	28.0
G223_M45_2																23.6%	51%	145 bp	18%	28.0
G223_M45_narrow_MACS2			235	45396
G223_M45_sorted_filtered	32.1	32.1			100%	225 bp	235 bp	1.19%	0.0	32.1	100.0%	100.0%	0.8%	32.1
G223_M46															78.4%
G223_M46_1																27.1%	51%	136 bp	0%	12.8
G223_M46_2																25.8%	51%	135 bp	9%	12.8
G223_M46_narrow_MACS2			218	38213
G223_M46_sorted_filtered	13.8	13.8			100%	208 bp	219 bp	1.81%	0.0	13.8	100.0%	100.0%	2.9%	13.8
G223_M47															81.5%
G223_M47_1																12.6%	49%	145 bp	9%	5.8
G223_M47_2																12.4%	49%	145 bp	9%	5.8
G223_M47_narrow_MACS2			243	22317
G223_M47_sorted_filtered	6.0	6.0			100%	235 bp	244 bp	1.99%	0.0	6.0	100.0%	100.0%	1.1%	6.0
G223_M48															82.4%
G223_M48_1																14.5%	53%	145 bp	18%	7.1
G223_M48_2																14.4%	53%	145 bp	18%	7.1
G223_M48_narrow_MACS2			234	25166
G223_M48_sorted_filtered	8.0	8.0			100%	226 bp	235 bp	1.87%	0.0	8.0	100.0%	100.0%	1.0%	8.0
G223_M49															75.5%
G223_M49_1																30.2%	48%	135 bp	9%	16.0
G223_M49_2																28.2%	49%	135 bp	9%	16.0
G223_M49_narrow_MACS2			190	48733
G223_M49_sorted_filtered	14.7	14.7			100%	182 bp	191 bp	1.84%	0.0	14.7	100.0%	100.0%	2.9%	14.7

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
\|\|	MACS2	Fragment Length	Fragment Length	`d`	None
\|\|	MACS2	Number of Peaks	Total number of peaks	`peak_count`	None
\|\|	Picard	% Aligned	Percent of aligned reads	`PCT_PF_READS_ALIGNED`	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
\|\|	Samtools	Error rate	Error rate: mismatches (NM) / bases mapped (CIGAR)	`error_rate`	None
\|\|	Samtools	M Non-Primary	Non-primary alignments (millions)	`non-primary_alignments`	read_count
\|\|	Samtools	M Reads Mapped	Reads Mapped in the bam file (millions)	`reads_mapped`	read_count
\|\|	Samtools	% Mapped	% Mapped Reads	`reads_mapped_percent`	None
\|\|	Samtools	% Proper Pairs	% Properly Paired Reads	`reads_properly_paired_percent`	None
\|\|	Samtools	% MapQ 0 Reads	% of Reads that are Ambiguously Placed (MapQ=0)	`reads_MQ0_percent`	None
\|\|	Samtools	M Total seqs	Total sequences in the bam file (millions)	`raw_total_sequences`	read_count
\|\|	Bowtie 2 / HiSAT2	% Aligned	overall alignment rate	`overall_alignment_rate`	None
\|\|	FastQC	% Dups	% Duplicate Reads	`percent_duplicates`	None
\|\|	FastQC	% GC	Average % GC Content	`percent_gc`	None
\|\|	FastQC	Read Length	Average Read 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

Picard

Picard is a set of Java command line tools for manipulating high-throughput sequencing data.

Alignment Summary

Please note that Picard's read counts are divided by two for paired-end data. Total bases (including unaligned) is not provided.

Mean read length

The mean read length of the set of reads examined.

Base Distribution

Plot shows the distribution of bases by cycle.

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

Insert Size

Plot shows the number of reads at a given insert size. Reads with different orientations are summed.

Mean Base Quality by Cycle

Plot shows the mean base quality by cycle.

This metric gives an overall snapshot of sequencing machine performance. For most types of sequencing data, the output is expected to show a slight reduction in overall base quality scores towards the end of each read.

Spikes in quality within reads are not expected and may indicate that technical problems occurred during sequencing.

Base Quality Distribution

Plot shows the count of each base quality score.

Samtools

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

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 Flagstat

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

Bowtie 2 / HiSAT2

Bowtie 2 and HISAT2 are fast and memory-efficient tools for aligning sequencing reads against a reference genome. Unfortunately both tools have identical log output by default, so it is impossible to distiguish which tool was used. .DOI: 10.1038/nmeth.1923; 10.1038/nmeth.3317; 10.1038/s41587-019-0201-4.

Paired-end alignments

This plot shows the number of reads aligning to the reference in different ways.

Please note that single mate alignment counts are halved to tally with pair counts properly.

There are 6 possible types of alignment:

PE mapped uniquely: Pair has only one occurence in the reference genome.
PE mapped discordantly uniquely: Pair has only one occurence but not in proper pair.
PE one mate mapped uniquely: One read of a pair has one occurence.
PE multimapped: Pair has multiple occurence.
PE one mate multimapped: One read of a pair has multiple occurence.
PE neither mate aligned: Pair has no occurence.

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.

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

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.

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

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.

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

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.

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

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.

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

Sequence Length Distribution

The distribution of fragment sizes (read lengths) found. See the FastQC help

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

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.

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

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.

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

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.

No samples found with any adapter contamination > 0.1%

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:

v1.12

MultiQC Toolbox

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Picard

Alignment Summary

Mean read length

Base Distribution

Insert Size

Mean Base Quality by Cycle

Base Quality Distribution

Samtools

Percent Mapped

Alignment metrics

Samtools Flagstat

Bowtie 2 / HiSAT2

Paired-end alignments

FastQC

Sequence Counts

Sequence Quality Histograms

Per Sequence Quality Scores

Per Base Sequence Content

Rollover for sample name

Per Sequence GC Content

Per Base N Content

Sequence Length Distribution

Sequence Duplication Levels

Overrepresented sequences

Adapter Content

Status Checks

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MultiQC Toolbox

Apply Highlight Samples

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Apply Show / Hide Samples

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Tool Citations

About MultiQC

General Statistics

General Statistics: Columns

Picard

Alignment Summary

Mean read length

Base Distribution

Insert Size

Mean Base Quality by Cycle Help

Base Quality Distribution

Samtools

Percent Mapped Help

Alignment metrics

Samtools Flagstat

Bowtie 2 / HiSAT2

Paired-end alignments Help

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

v1.12

Highlight Samples

Rename Samples

Show / Hide Samples

Mean Base Quality by Cycle

Percent Mapped

Paired-end alignments

Sequence Counts

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