European Genome-Phenome Archive

File Quality

File InformationEGAF00000643571

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.

194 552 97436 407 6049 796 3216 068 8434 452 3093 762 5923 303 8322 986 9282 738 5212 547 1132 376 4112 237 4452 115 6762 021 8921 931 8541 855 9741 785 3391 720 8801 657 3051 604 9491 547 6421 502 1351 460 4541 418 4821 377 1591 338 4311 302 6531 264 6091 231 0521 195 8971 157 3581 120 7371 087 7051 055 3911 020 061987 389957 549926 101895 853864 210838 861811 685782 238755 836730 456706 907683 764657 747634 364609 183586 312564 946545 337526 172509 106490 278473 348457 111438 660423 504409 061394 853380 222368 220355 925342 064329 832318 256307 943297 703288 656279 370270 370261 146252 039242 931233 924227 043220 449212 245205 271198 534191 856186 874180 853175 637169 974164 540159 319153 950149 734144 923140 921136 711132 548127 911125 038121 473118 949114 017110 920108 121104 938102 30299 31696 55294 10291 73988 78786 49483 79781 23979 90677 59876 54873 89771 80669 50168 30765 98464 40062 86960 73159 90657 85156 79655 21153 50251 66050 84249 72348 55647 51846 17744 79044 17543 09842 07941 20639 79239 01438 12537 46536 21435 70834 69333 97633 48032 80731 87131 44430 72029 83729 03828 40327 59327 00526 53925 93025 75424 88424 56223 86823 27222 86922 82122 33821 54621 23420 79320 10119 89819 45318 96518 40417 99017 60217 42117 07216 59116 37515 74715 49715 26314 97914 47814 23214 33313 83913 64913 38612 90112 81012 69912 03711 74611 89911 49311 21010 88010 80110 42110 23510 2549 8879 7629 4489 2469 0898 9559 0708 8008 4328 3108 1617 9007 8557 5857 4727 3997 3526 9587 1056 7646 6276 6156 5506 4066 1576 1625 8945 9925 8015 6365 5835 4155 4905 4775 2785 1134 9564 9054 9754 7944 6724 6934 6164 5424 5134 2974 2154 2614 1363 9873 9973 8083 7183 6253 6373 6643 4723 5683 4773 5283 4283 3843 3723 2933 2313 2583 2213 0133 0053 0883 0372 8822 9482 9222 8752 9452 8322 7872 8262 7402 6962 5452 5442 4782 4562 4792 4132 2802 3052 2562 2872 2282 2722 1982 2392 2672 2652 1852 1392 0922 1032 0472 0351 9581 9901 9061 9081 9111 9181 8861 8461 7911 7071 7171 7071 6291 6601 5951 6011 5891 5991 6281 5651 5571 5891 5151 5281 5151 5221 4441 4241 3821 3961 3981 4121 4281 4581 3301 3101 3201 3231 3641 2921 3061 3111 3491 3401 2431 1961 2781 2051 2781 2121 1901 1511 1891 2081 1281 0921 1691 0781 0841 0421 0991 0781 0721 0611 0681 0601 0299991 0401 0229959659751 0089751 00499894795896999297897394889196694495286990884987982684385581374978380376678578276074974274777977280172976578274874375374774374974176271767968064067464966564461869266964161559568567159161356359760858258157255054057356653648647951752046147945548847351348146950247249351347543247248945943548546648846046946549044747250743346941545543941440440843640539438339838540336639536835136537936739640136035033734835639234834836033735030932533930731433034531031327530530230832630631933729232034529535830929029331131428631429028529729128629628327328627028330327824924030029827526628725725025028427925825626727125426226925227624225023523827427726123126625426224523826026124925624726924924624025121026321823923225823423523023827023921225424022325522420818922820620924021820522122023021621222721523923822523021722521320221020921719217821121219719821420720820620118719018520319618720820619821223817718818319419819818219319716418318817720019318619719018218018518618517017317217219616017216416016515417016816617015215417016213614013913114512913415014014711813212712811612513115114114714313715915214214812614412715013412415112911313712913212210910911911312012112111811512711012011511012411310211611010710212110310910810512011210210810010911710811399949410394100102971109710999108928910010098638478899294977492918388829395799199777974677877928274937382817175727785758474888276908771787492877983937458696156745474637179657769816763495765556959696078806075736258726664716157535954785267615963595559596364535777665860747061726754485865544650416262494947574754554448485547455539513643464250454247453230564435334552343833374142393551355251394334505352384758504346563648464141384510 833100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

349 704248 899282 006645 022550 463416 5565 124 8572 321 6171 760 31417 479 98814 862 04910 485 6113 253 4211 368 8815 809 3175 102 1612 019 72913 774 10936 485 22427 168 41114 895 05318 233 24416 228 96613 458 7409 055 99411 056 11034 668 50749 167 12850 475 91241 857 71742 828 60845 809 73971 938 22769 131 74789 807 241149 517 045197 658 301199 834 781184 429 135288 691 487346 472 442364 641 310520 795 415374 689 573125 380 88764 777 78020 072 8788 193 5204 549 6740051015202530354045Phred quality score0M50M100M150M200M250M300M350M400M450M500M# 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).

99.2 %47 316 31499.2 %0.8 %

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.

98.9 %47 161 70698.9 %1.1 %

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%).

0.3 %154 6080.3 %99.7 %

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 %23 852 17050 %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.

98.7 %47 101 99298.7 %1.3 %

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.

6.1 %2 904 3416.1 %93.9 %

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.

5 315 4454 0103 4307 2504 40610 7649 42519 84716 57647 46040 63011 41389 60916 03615 026199 03535 50198 82271 92026 940143 0941 22490 887315 6111 14618 7581 6031 5771 8301 339 7564 4213 6605 0306 1326 2828 866198 474586 01817 2444 67230 26816 2801 85244 1221 8043 898123 7563 3347 5925 47015 9247 12025 03025 01039 25673 722178 31838 301 754051015202530354045505560Phred quality score5M10M15M20M25M30M35M# 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.

99.63%99.66%99.79%99.49%99.76%99.8%99.6%99.75%99.46%99.52%99.79%99.84%99.8%99.81%99.59%99.55%99.67%99.75%99.8%99.78%99.61%99.66%95.46%99.61%0.37%0.34%0.21%0.51%0.24%0.2%0.4%0.25%0.54%0.48%0.21%0.16%0.2%0.19%0.41%0.45%0.33%0.25%0.2%0.22%0.39%0.34%4.54%0.39%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped