European Genome-Phenome Archive

File Quality

File InformationEGAF00002240169

File Data

Base Coverage Distribution

This chart represents the base coverage distribution along the reference file. Y-axis represents the number of times a position in the reference file is covered. The x-axis represents the range of the values for the coverage.

Data is represented in a log scale to minimise the variability. A high peak in the beginning (low coverage) and a curve descending is expected.

65 299 904140 709 743236 664 844329 217 390391 109 413404 731 969371 118 627305 494 741228 982 293157 742 491100 937 80860 603 68234 494 33618 830 36210 066 5805 352 6462 953 7481 761 4801 145 094829 889654 432545 657470 663409 831360 606324 556291 326263 694239 504220 861202 022184 763173 712161 419149 593140 654130 142121 309111 065101 89794 76188 06780 99673 29368 30762 24757 43254 81651 15846 73744 28441 34739 69837 90135 75633 23632 83930 71528 85528 42026 86826 25824 69924 03222 51322 60121 92121 00419 93720 20619 12918 42317 67816 96816 38316 52415 65415 54714 41713 83013 69413 72213 44513 14412 52112 34912 00811 78711 35911 19810 76010 77710 53110 3259 9669 9199 8799 6949 1799 5159 5228 8618 6868 6098 6018 4088 2228 2247 9167 6787 8407 5987 1777 3246 9366 9476 7846 6956 4476 5776 5996 5076 1296 0216 1075 8475 9525 9405 8085 8115 6005 4245 5685 5695 3665 4375 2705 2675 1954 8764 9434 7994 7834 6714 7254 7354 6184 6064 6334 4804 5574 6234 3114 4204 3074 1713 9704 0434 0114 0533 9613 9233 8393 9164 0543 9143 7253 6963 6213 5863 4693 5143 4323 4283 3513 4563 2323 1363 2513 2023 3303 2353 1213 0302 8872 9902 9243 0052 8922 9312 6732 6982 6672 7522 5512 7922 6432 5442 5372 5522 5472 6122 4982 3342 4122 4572 3642 3242 1932 2132 1742 2272 2762 2622 2992 1392 1982 0492 0312 0271 9392 0192 1222 0592 1021 9652 0321 9811 9311 9441 9741 7981 8431 7331 6801 7301 6661 7431 7781 8491 7491 5491 6721 6431 6301 6191 6231 6071 6731 5561 5961 6281 6071 6101 4841 6361 4501 5811 5341 4561 5581 5281 5331 5361 5451 5001 5611 5201 5231 5051 4871 6421 4611 5031 4371 5081 4851 4581 4141 4371 4681 4191 3331 3391 3551 3081 3421 4581 3851 3231 3671 3781 4371 3671 4551 4491 5551 5101 5591 4761 3661 3611 2981 4001 3341 3301 2921 2771 3081 2811 2671 2441 2601 2611 2441 3001 3001 2381 2501 2391 1801 1531 1911 1391 2021 0581 1521 2421 2261 1611 1361 1641 2091 2551 1911 2101 1331 1131 1461 0761 1501 0731 0101 0401 0611 0631 1201 0489981 0841 0859659599829799519871 0039791 0549989619661 0221 1011 0361 0401 0011 0221 0049729559399349281 011968969942963942927919897964907961915899929896827829853894883884910902872831826850861807844928856852845865882831844860864825843780773781769759783771705775753698767707721720733715707703687737669680668684640682701703631609671650606613584623601581688656679633685645608586567571546583522499561532557529517528488487487488539455482464480473511450474482487507500444493497438500467442468481443440471455491475475429434436417424424456420399440439422432448457412408411420441442420444431429354401418430413409407401434452469452418464475407396378405414421412418400387374347372346356305350346337340393380361374369351326336348374347347287323323317349360339343327335338348339341358333308322310308310313305292311314318307300355325290282310316317317309286312318337275298304310313321344323335322292319300343337320325315310297327303303312326311322303308288267277284274271293320286295273265325285264300295290277307261288289284262270260307259264276301229299255272269315292280273302272321295288289308320305290329279292310293308285279309290255257302292313295316325294252263293305280309278257281294286281261299264267293285274291273288248253260241252262240282271305294307306284267278293306310305276298340318295332348321348333353321325337327310345326317306312308295304290315273307278309277280264296297294318299299305300286277295257258275244262293275268254228270281273265249269279236244261250268278232276252264229251231252242247212236215205224221200188221221198213217181202210210200188173180190203204183189182181201189185167154182181201218181177183151181205173188163187165166189177189189191177185170171167165170174142154170175140148161166183172165146140156166154160169159142141145163144152149173175183177178161153145187148181175174180164142175162181181204189171189167176182181154185172201168160204194183205172170201161193180180193191166203172187190172173191231207185209194195202194216191235197210217212133 308100200300400500600700800900>1000Coverage value1k10k100k1M10M100M# Bases

Base Quality

The base quality distribution shows the Phred quality scores describing the probability that a nucleotide has been incorrectly assigned; e.g. an error in the sequencing. Specifically, Q=-log10(P), where Q is the Phred score and P is the probability the nucleotide is wrong. The larger the score, the more confident we are in the base call. Depending on the sequencing technology, we can expect to see different distributions, but we expect to see a distribution skewed towards larger (more confident) scores; typically around 40.

1 543 374000000056 216 888000926 206 062000000000555 293 5750000657 191 90200001 343 523 34800002 722 632 29400012 882 242 54300510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G12G# Bases

Mapped Reads

Number of reads successfully mapped (singletons & both mates) to the reference genome in the sample. Genetic variation, in particular structural variants, ensure that every sequenced sample is genetically different from the reference genome it was aligned to. Small differences against the reference are accepted, but, for more significant variation, the read can fail to be placed. Therefore, it is not expected that the mapped reads rate will hit 100%, but it is supposed to be high (usually >90%). Calculations are made taking into account the proportion of mapped reads against the total number of reads (mapped/mapped+unmapped).

99.8 %126 478 85199.8 %0.2 %

Both Mates Mapped

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. This chart shows the fraction of reads in pairs where both of the mates successfully map to the reference genome. .

Notice that reads not mapped to the expected distance are also included as occurs with the proper pairs chart.

99.6 %126 234 39299.6 %0.4 %

Singletons

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. If one mate in the pair successfully maps to the reference genome, but the other is unmapped, the mapped mate is a singleton. One way in which a singleton could occur would be if the sample has a large insertion compared with the reference genome; one mate can fall in sequence flanking the insertion and will be mapped, but the other falls in the inserted sequence and so cannot map to the reference genome. There are unlikely to many such structural variants in the sample, or sequencing errors that would cause a read not to be able to map. Consequently, the singleton rate is expected to be very low (<1%).

0.2 %244 4590.2 %99.8 %

Forward Strand

Fraction of reads mapped to the forward DNA strand. The general expectation is that the DNA library preparation step will generate DNA from the forward and reverse strands in equal amounts so after mapping the reads to the reference genome, approximately 50% of them will consequently map to the forward strand. Deviations from the 50%, may be due to problems with the library preparation step.

50 %63 393 54350 %50 %

Proper Pairs

A fragment consisting of two mates is called a proper pair if both mates map to the reference genome at the expected distance according to the reference genome. In particular, if the DNA library consists of fragments ~500 base pairs in length, and 100 base pair reads are sequenced from either end, the expectation would be that the two reads map to the reference genome separated by ~300 base pairs. If the sequenced sample contains large structural variants, e.g. a large insertion, where we expect the reads mapping with a large separation would be a signal for this variant, and the reads would not be considered as proper pairs. Based on the sequencing technology, there is also an expectation of the orientation of each read in the fragment.

The rate of proper pairs is expected to be well over 90%; even if the mapping rate itself is low as a result of bacterial contamination, for example.

98.5 %124 914 82098.5 %1.5 %

Duplicates

PCR duplicates are two (or more) reads that originate from the same DNA fragment. When sequencing data is analyzed, it is assumed that each observation (i.e. each read) is independent; an assumption that fails in the presence of duplicate reads. Typically, algorithms look for reads that map to the same genomic coordinate, and whose mates also map to identical genomic coordinates. It is important to note that as the sequencing depth increases, more reads are sampled from the DNA library, and consequently it is increasingly likely that duplicate reads will be sampled. As a result, the true duplicate rate is not independent of the depth, and they should both be considered when looking at the duplicate rate. Additionally, as the sequencing depth in increases, it is also increasingly likely that reads will map to the same location and be marked as duplicates, even when they are not. As such, as the sequencing depth approaches and surpasses the read length, the duplicate rate starts to become less indicative of problems.

3.6 %4 509 3253.6 %96.4 %

Mapping Quality Distribution

The mapping quality distribution shows the Phred quality scores describing the probability that a read does not map to the location that it has been assigned to (specifically, Q=-log10(P), where Q is the Phred score and P is the probability the read is in the wrong location). So the larger the score, the higher the quality of the mapping. Some scores have a specific meaning, e.g. a score of 0 means that the read could map equally to multiple places in the reference genome. The majority of reads should be well mapped, and so we expect to see this distribution heavily skewed to a significant value (typically around 60). It is not unusual to see some scores around zero. Reads originating from repetitive elements in the genome will plausibly map to multiple locations.

4 292 24187 07950 735106 33274 46579 20292 395121 52855 89399 18642 97237 56156 50761 20232 34178 52152 25760 03876 86799 381100 585104 866126 34297 274163 999281 13016 227566 56023 80323 33455 34749 22422 43063 11225 10025 02343 91056 16514 97289 2221 468 15866 61261 345104 22187 668163 870149 016208 608386 98839 20255 97647 02366 72831 50054 16856 96140 413160 84642 77688 235116 098 849051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M# Reads

Mapped vs Unmapped

Stacked column chart for both mapped and unmapped reads along all chromosomes in the reference file. It is a similar representation as shown in the Mapped reads chart but for each chromosome. Although sequenced sample may be a female, it is possible to get reads in the Y chromosome as there are common regions in both chromosomes called pseudoautosomal regions (PAR1, PAR2).

Unmapped reads belonging to each chromosome are determined when the one mate/pair is aligned and the other is not. The unmapped read should have chromosome and POS identical to its mate. It could also be due when aligning is performed with bwa as it concatenates all the reference sequences together, so if a read hangs off of one reference onto another, it will be given the right chromosome and position, but it also be classified as unmapped.

99.8%99.8%99.81%99.81%99.81%99.81%99.81%99.8%99.8%99.79%99.8%99.81%99.81%99.8%99.8%99.8%99.8%99.8%99.8%99.79%99.8%99.81%99.85%99.72%0.2%0.2%0.19%0.19%0.19%0.19%0.19%0.2%0.2%0.21%0.2%0.19%0.19%0.2%0.2%0.2%0.2%0.2%0.2%0.21%0.2%0.19%0.15%0.28%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped