European Genome-Phenome Archive

File Quality

File InformationEGAF00002047643

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.

27 272 30962 586 874116 160 847179 731 686239 872 273284 585 470306 543 318304 446 423282 278 110246 963 963205 538 679163 877 648125 780 42693 533 70067 737 73247 875 25633 283 06322 702 12015 354 37910 353 8136 955 1114 705 2163 191 7352 211 1491 572 8621 152 536862 444668 965542 163450 181379 332327 572290 227258 043236 255214 450196 081181 043169 899157 789146 872140 320130 495123 324116 900111 457105 48098 22294 06288 51284 04578 38075 16571 16668 24064 95960 91257 10854 82252 03649 87846 10244 31342 32139 91938 14936 14334 69832 51231 31129 87829 57927 87726 56525 85524 23023 36622 38120 72520 55419 72719 00218 41718 21817 81017 11716 61015 96215 86615 32414 88514 58114 10214 17613 90712 81313 05412 86412 55412 02111 51911 10511 42910 79910 39710 62310 48210 2029 92810 0009 5799 2399 1019 4039 4568 9008 8088 8878 5028 5828 3028 2017 9587 9617 5577 4687 4597 3937 3007 2736 8626 7926 7336 4466 3136 3566 0156 1126 2576 0296 0025 9525 9655 6215 5215 3735 3065 0915 0745 0805 0094 8954 7124 6984 6644 6254 6334 5164 4744 4754 3864 4624 4434 3794 4704 5504 3044 3374 2724 0704 1473 9783 9713 9053 8963 6613 7793 8773 7153 5963 4733 5453 5983 4163 3833 3033 3073 2923 3323 0863 1603 0242 9372 8652 8762 9302 7412 8102 7612 8662 7382 7822 8602 8912 8622 6582 6672 7352 6912 6682 7072 7742 5542 5282 5912 5022 5372 5222 5942 4382 5342 5952 5712 4092 3162 3542 3862 1882 3192 2292 3772 3172 4272 1472 1282 0372 2792 1852 2802 1062 1802 1882 1682 1062 0742 1212 0641 9681 9751 9871 9231 8291 9391 8791 9081 8411 7571 8331 8791 8891 8741 7191 6361 7421 7121 7201 7631 7521 6891 6221 5931 5031 5921 5871 5791 5561 5371 5851 4821 5401 4371 4431 4861 4361 4531 4661 4701 3461 4441 4271 2961 2741 3131 2851 2451 2741 3221 2761 2461 2471 2821 2281 2901 2661 2411 1931 1871 3251 1861 2041 2231 2321 2471 2511 1651 1991 1881 1281 1361 2601 2191 1921 1571 1821 1791 0901 0931 0891 1581 1141 0981 1541 0911 0801 1561 1091 1281 1061 1121 0591 1029961 0181 0739999801 0819931 0731 0431 0149249669829489239839881 0221 0051 0079919891 0329829649859698968999479298788819499219229648879259529581 0109281 0021 040928950935856913870847836860850936982877829865940923910910829847887851884848853856925888884860845865798830819800773808782810792770793798750843817920848829813766810823875828707792763782829760748769749787764724731723715713696730700734745678678713735701666655621684689648631636688600632658630663637609640615599623642622606633623594601579652616556612560608569545550567606528547549531524578559578558577532564569548576593547607631598546594607571551547569524557539505533507490529486522492520623439449454499422469444451447415432487449453404442421480442445472468476442438451450450434431507471483461421415373368438405460429403392383408413389410384366395389372384395431394384490389374354390375373329386353380391373365383359353378396357317349353365329325381330404357338324354343342322347320316335334319297331338295322340338298342328334338321332360352355371320353321361363338329344357323330319307305339331312296334297314282327318347340300307308288317299311262291352334285316279304289311316344347253311310320323291297320290311296347345302327298291333273334304325326335353335304336319325296247311268311287281294292317311316269283278285289297275289290281282292270317272290273277320269282288274290286278265282255257260307248282274288274283291275248276286261261259258257254274256286265274284291309270284279250252272240264266249251264266283272229267227254279251246241246304276287263252249259234252266304238253271255272272285270274299307258264283261268253273272259256237292255258253243246243239259258283264270263289287281266267295250278286259246249308250221235263275273254258256253217237228241216246249251243247247242264262265225225241227252242223228236244259251284271253256265255252266264284276275259274268268249244271269280244287253269243240273309289291274276264249236286253243237286288274304300259281231261247292237242278221222236213243210206240243239228231243291 907100200300400500600700800900>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.

1 470 7700000000128 682 7970001 345 837 228000000000792 967 2850000903 311 79800001 790 230 98200003 565 266 21300017 680 819 12300510152025303540Phred 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 %172 312 58599.3 %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.

99.1 %171 978 29099.1 %0.9 %

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 %334 2950.2 %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 %86 783 39850 %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.

97.8 %169 723 57897.8 %2.2 %

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.

15.5 %26 834 53015.5 %84.5 %

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.

7 758 614140 84486 635168 155122 735127 215146 014194 77689 809159 26171 38161 43188 554101 52254 017122 09781 04588 968124 892175 576179 774177 748232 262177 547286 010469 20527 543819 83939 97638 60976 40980 31236 479101 96141 10941 44966 98586 54724 202134 3842 000 189100 07397 653162 872141 561261 776225 215362 185562 80164 81583 53172 33794 62745 89987 20685 08163 658230 71967 740135 322156 765 221051015202530354045505560Phred quality score20M40M60M80M100M120M140M# 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.

99.81%99.77%99.8%99.8%99.8%99.81%99.81%99.8%99.81%99.8%99.81%99.81%99.81%99.8%99.8%99.82%99.82%99.8%99.83%99.8%99.81%99.8%99.58%99.77%0.19%0.23%0.2%0.2%0.2%0.19%0.19%0.2%0.19%0.2%0.19%0.19%0.19%0.2%0.2%0.18%0.18%0.2%0.17%0.2%0.19%0.2%0.42%0.23%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped