European Genome-Phenome Archive

File Quality

File InformationEGAF00008413801

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.

37 916 00777 799 421131 416 471192 031 543248 865 777291 056 881311 123 759307 613 708283 794 338246 275 651202 467 201158 671 978119 043 38986 040 58060 241 57740 943 17127 247 06117 738 51111 455 0947 359 2474 740 3833 120 2962 108 6091 485 4401 091 293837 448672 411558 890477 104413 771369 838329 525299 721273 399250 518229 616213 391197 186184 532170 611160 133151 343140 662133 635125 949117 008111 091105 46299 85294 13487 21882 34777 63973 09868 97663 65961 21656 79152 30649 04247 14145 30943 08240 04138 38236 48934 96933 62932 88631 10929 93228 75527 48726 97425 70524 65824 31823 24822 57122 17720 95520 31920 06919 67619 12018 18518 18217 25017 04816 62516 35416 01715 67315 33714 55614 48014 24913 83014 17913 70413 08112 90112 84912 50511 54011 47111 49111 36011 05610 92210 34810 29510 31910 1239 8839 9169 7789 4359 3489 0359 0758 7148 7428 5068 5908 1608 1877 9257 6877 6867 7887 4007 8397 3897 4197 2787 4127 2787 0907 1127 0676 6997 1626 7596 6296 5716 4586 4576 3776 0856 2026 0006 0976 0825 8325 8135 8605 7945 8725 8315 6485 3655 4435 3745 2254 9875 2185 1315 1634 8334 7274 9324 8144 7154 5424 7714 6894 6284 5334 2994 5204 2244 2284 2554 2024 1174 0464 0413 8833 9053 8383 9713 9563 9523 9203 8273 7223 6833 5843 6243 6273 4443 4743 5183 5023 3393 3713 1743 3173 2213 1793 1613 1033 2953 2123 1033 0763 0072 8452 9782 7862 8412 7592 7042 8402 6652 5782 6312 6372 5482 5442 5952 5662 6962 6712 6002 4182 6212 4242 4332 3582 5142 4502 4372 3352 4852 3232 3872 5282 4002 4432 4452 2372 3362 2322 2632 1512 1602 1082 0452 0861 9831 9652 0111 9711 9191 9121 9511 8941 9111 9421 9241 8971 8861 8971 8681 8841 8871 9191 8391 8941 8141 8441 7801 7721 7391 7101 6381 6461 6551 7031 7311 6911 6141 6741 6391 6061 6351 5801 5121 5081 5121 5261 6041 5431 5731 4941 4241 4901 4901 3791 3851 4641 4021 4251 3671 4361 4431 4181 5141 4041 4221 4091 3931 3461 3191 3721 3671 4511 4161 3371 3701 3701 3011 3781 3301 3241 3221 3571 3371 4171 3421 2951 3241 3271 2941 2471 1881 2271 2141 1291 1471 2071 2531 1881 2181 2851 1841 2051 1371 1821 1401 1571 1261 1371 1161 0901 1371 1951 1731 1121 1241 0361 1041 1041 0811 1011 0911 1231 0101 1201 1091 0749911 0191 0371 0881 0381 0291 0501 0569421 0359981 0079619981 0771 0041 0241 0591 0301 0041 0411 0429851 0469611 0569389911 0589699419441 032979992938948898921907938940863910910850840862898838865862874874922835831898836878846869877898800830823838884883825818837827802781855776822832849791841739797863826888810804810868859800809847833793780766760806757781669762726705721705724669689768731700700679656699695676744693671683653649685633636684654670665629655643596624609654611638621637650623634662566672619634601638609661610610577619589629549564563607641585651644673632549593645600633595546615584581580599593571599563579554555540586579537512510519491510548536493498498526520471541557530532535503579526523537568509557522570539515536495500492485490471478536504479465439468466446444485450522439497455415426424452456424448430446419480430483454498445442420429436406445434416463427395479428459490496421388381410401364369418368429373382379348357403407362372390382380401381393379399375372391399360384359392421380384382371397375375371383388384375362382391404381371405358368346338365387377415351379395354348403375376432395366360395409377389374386384364388356348383380351377371367379367392483438376370354397371367363347386410406390425368367365346372345340386349397364339351397363338372387331366349343341351333367322344321328340366350355340312307370366347328361339334340337343333350330354352356344375369325335365343354374369354365350372368378347319324320329352304348382317314328337328299333353336345361326336313346307337327364345313306317328303307325325322326345287324313322328317315328330287294288280279299305307304313287311327318332304309319290278316286295300293289309312292314323328320304279292277299282297283304310299314330308320333300320315331297296278271303278299341262314303285304280279270283295268258267276259295294301272286292261304297275307273272293281267271290290317303272269303307294271259305301308314 073100200300400500600700800900>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.

3 812 588000000061 261 094000790 954 066000000000479 026 0490000599 660 48500001 360 797 63100002 915 788 08200019 150 267 70700510152025303540Phred quality score0G2G4G6G8G10G12G14G16G18G# 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 %167 666 15499.8 %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.

99.8 %167 541 78299.8 %0.2 %

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%).

0.1 %124 3720.1 %99.9 %

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 %83 978 70150 %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.

98.3 %165 111 96498.3 %1.7 %

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.

13.5 %22 742 09913.5 %86.5 %

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.

7 136 939146 83388 234174 671130 363137 688144 655212 22296 002158 92674 16664 35486 138105 12954 180124 09991 274100 436122 708177 826188 093179 607232 854185 457292 974499 62126 482830 43240 25038 68874 59783 69238 033100 79140 75639 71162 38989 60423 041135 5321 986 75692 98683 740155 422127 721248 777211 558331 363552 24254 49276 48063 72386 17735 72968 950106 72148 489214 26650 610115 346151 836 037051015202530354045505560Phred 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.

99.92%99.92%99.92%99.92%99.92%99.92%99.93%99.92%99.92%99.92%99.93%99.92%99.92%99.93%99.93%99.94%99.93%99.92%99.93%99.93%99.92%99.92%99.95%99.93%0.08%0.08%0.08%0.08%0.08%0.08%0.07%0.08%0.08%0.08%0.07%0.08%0.08%0.07%0.07%0.06%0.07%0.08%0.07%0.07%0.08%0.08%0.05%0.07%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped