European Genome-Phenome Archive

File Quality

File InformationEGAF00002308093

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.

43 480 08693 891 552162 528 139238 541 669304 980 785346 142 117353 919 719329 387 388282 353 238224 689 498167 634 591118 049 59878 996 51050 602 02831 227 06418 725 86511 033 7236 499 0353 892 1092 434 1991 605 9451 147 127878 008714 279606 897523 209455 855406 988366 084330 265298 076269 718248 712224 802207 125189 673175 182163 455153 727141 201131 743122 619114 843106 50599 50293 35987 67383 08577 26972 19967 67164 10860 57656 89054 09251 38247 73446 27743 02941 15539 44237 98435 61334 22033 34832 13731 10829 85527 90027 54626 80625 86724 84524 50623 66722 69321 96820 99020 32019 97919 82518 44818 11918 43617 25616 98916 27216 00615 52815 46114 70914 41913 81113 46713 30013 02312 38612 43612 61111 99611 82011 82011 30010 96110 95810 93910 91410 6979 99110 1699 9109 6839 4859 3669 2459 2148 9548 9518 6698 6428 2468 0187 9467 6767 5557 7037 2727 4717 2137 4247 2427 1296 9567 1757 0906 8086 9086 7946 5696 5556 6436 2736 1606 0006 1225 8085 9985 8055 5765 4855 4985 2805 2775 1685 1795 1875 0504 8264 6294 5774 3524 4334 5164 3774 2874 4064 4084 2684 1374 3334 2414 1794 1684 0554 0224 0653 9083 7993 9113 8063 6413 6393 8763 5183 4053 4303 4503 5473 5383 2443 2223 1303 1383 2293 1203 0613 1673 0762 9893 0192 9792 8823 0152 9913 0493 1633 0023 0813 0522 8722 9152 9952 8972 8432 9222 9092 7242 7502 5482 6392 6432 5912 4462 4902 5922 6312 3552 4752 4032 3512 4352 5082 4382 4942 4482 3502 3182 3112 3942 4152 3412 3422 2682 2712 3372 2332 2152 2422 1872 2372 0952 1382 0532 0082 0472 0841 9881 9612 0602 0302 0051 9691 8791 9231 9341 9422 0251 9351 9912 0231 9691 9351 9862 0611 8791 8511 8281 8341 8481 7641 8711 8471 8001 8591 7271 6891 7431 7091 7411 7821 7171 6871 5891 5901 6401 6191 5341 6511 6001 6601 5601 5261 5901 5211 6151 5471 5101 5071 5841 5701 4571 5251 5281 5611 3741 4281 3751 4531 3521 4721 3481 4271 4181 3581 3631 3051 2491 2701 2401 2541 3321 3371 3091 3561 3511 2561 2541 1921 1891 2871 1891 1851 1651 2061 1461 1821 1591 1901 1731 0991 1341 1241 0521 1341 1111 1141 0481 0841 0851 0501 0641 0891 0291 0741 1191 0541 0641 0561 0341 0731 0751 0601 0219171 0629631 0591 0481 0129531 0081 0059661 0271 0039639499729199029931 0009851 000910946893834866857835851857881836896822858890869856899919891883805824828783878784869791859895821826845810814783837801761741747735704696728761808753768790737814749812760767743728751693761741725784742749734727735699700701707689694755714715687703718703730715694675642684690682638716690656619684649670644653626638629596626617649681635625601590615643627609592631630554616548604605598552634575572641648612614581588621560594594556598560595581565557580586573544606587569547562591596570580647664604575576620614618570603605612608640571553513579563549520577558540575544538522539563522580552556570560532546550537497583539534541522557569578572533547591551570563537539541559548537542512505565550551554513562576508547512516556511509502489560563529502508556536549499526501477518531533537482516474497547506469444502474434452539494463441430424469455452446444484429468441482454451460433455418427418449458421484524494437432457438469416460386437390421408438385378383425392428425411423431468448429479500479451469464438455471428421388460478450409457415463446411459413376409403429407457413382424433407444400375409371365407382341335382352370316360304322340337289312317320345310309305299315322306332285301322271288295268283310305315306290309324270276289321306286322322289299288286305296299271292316309313310309302270314281310290296308302259269256310266263264267253289266305268274273287280296267293265233271256294257236260284293294259281272260282290263304247293271299267280278262246275252250285255258285253244278254256291255270253263256283276282238287293231251263275265276265286282243285245269259268276265271264258299254272255269240267233281265260258221274265247219250235256270243217256249253243264247281253240255257218242239218219220233222210228239233202230228216266210251234241209226237206210236228241221206217221221212209200196199218209208197268 136100200300400500600700800900>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.

4 868 769000000024 031 017000999 755 873000000000563 417 0580000626 148 00600001 347 318 57200002 825 122 20700016 548 638 49200510152025303540Phred quality score0G2G4G6G8G10G12G14G16G# 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).

99.3 %150 790 93599.3 %0.7 %

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 %150 231 11498.9 %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%).

0.4 %559 8210.4 %99.6 %

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 %75 957 94750 %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.

97.4 %148 003 46497.4 %2.6 %

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.

8.8 %13 430 5618.8 %91.2 %

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.

7 133 890140 61584 819168 638125 916132 595154 632203 72489 555153 07071 47861 76092 92897 72550 250116 82982 61993 274121 762162 840172 389165 004199 609150 626250 924412 22425 682748 48838 24936 75683 44876 70136 59194 78539 63539 09469 14286 41723 151131 6461 975 20590 09887 654142 814120 093222 666194 632286 502496 49156 85876 15267 64487 98943 39680 11177 75056 761209 37358 497114 036135 856 009051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.61%99.6%99.62%99.62%99.62%99.62%99.62%99.62%99.61%99.61%99.61%99.62%99.61%99.61%99.62%99.64%99.64%99.6%99.66%99.6%99.62%99.62%99.75%99.51%0.39%0.4%0.38%0.38%0.38%0.38%0.38%0.38%0.39%0.39%0.39%0.38%0.39%0.39%0.38%0.36%0.36%0.4%0.34%0.4%0.38%0.38%0.25%0.49%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped