European Genome-Phenome Archive

File Quality

File InformationEGAF00002339607

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.

356 925 580478 366 001497 843 159437 872 988339 615 287239 233 020155 866 53995 414 71955 504 96831 100 12716 947 5739 115 3054 912 7312 734 7571 590 4271 004 364694 728508 775399 341332 973281 737239 057207 323183 321162 188145 637129 369116 928106 18896 96686 88180 16275 12768 94063 35059 81757 29654 40250 88149 21045 94343 58442 32840 83637 85036 25934 94734 17833 51331 53530 61928 51927 37725 83425 47424 36023 95423 39422 75121 88920 93220 52720 07619 62619 30218 26817 61416 84616 55816 56916 42415 61015 49914 48514 05114 04113 88613 37012 95712 91312 45812 52311 75911 65910 91110 51210 30610 47110 28710 1409 5129 4879 1739 5078 7698 6108 3178 4138 1688 0037 8237 6367 7857 5067 5347 1996 9646 8696 6046 4926 5096 6726 3466 1765 7795 8655 8275 8155 7585 6815 7385 4735 3795 1985 0485 0795 2115 1525 0974 8024 7875 1525 0754 7614 7984 8114 5954 3694 3614 4114 3434 2454 2913 9733 9073 9803 8213 7893 8673 9083 6893 6453 7423 6353 5913 4113 2783 3493 2263 1303 1843 1673 1483 1503 1283 1593 0883 2443 1112 9602 9102 8862 9702 7992 8742 8062 9952 6982 6102 6482 6992 6712 5792 5002 4872 4872 3212 3432 4962 4742 4192 3942 3182 3282 4642 3252 2492 2262 2142 3142 3332 2872 2532 2942 1682 2572 2352 0662 1522 1622 0852 1602 1231 9272 0172 0111 9612 1751 9371 9921 8641 8891 8691 8111 8261 7291 7001 6281 6111 6121 8171 7111 8241 8101 7581 7181 6741 7491 6611 6841 7181 6081 6931 7361 6661 5211 6521 5231 4511 4691 4511 4291 4331 4361 5261 4701 4021 5311 3611 4341 4101 4441 4941 5151 2921 3691 4301 3421 3411 3121 3711 3751 3251 2351 2161 2501 1631 2671 2071 1801 1591 1001 0891 1581 2451 1571 2581 1151 2161 2191 1721 2881 1751 1281 1211 1681 1171 0511 0839971 0181 0101 0209799981 0581 0611 0311 0028971 0039869889439579831 0079491 0109759971 0591 01793795692992488190484891593687184583288881082879988291284786889483479480683082686283786679172279479281384067870371978474969979077395572675077773076975985475971179777774374174478776372768878775473071462075168575371364067368474563971275467867074166871364266267968263361560160961159464260656356556663863657456563959560963847749356651651354055054950661952649651850548746451649450652151846045958253246544350044749351745051645645946951359553259261352249048346146943746845539737741039542438138842440341042741238344336745143637638343840943548242840437236936339741436931940237833738434637334634433731832532045740232129829430931431731234134032138030636229733940838433031634230133031733234635432634432337933834637735833133033531432331329530732333034031130133731128326829028230329031031831331430831529929434229830130430230726828426229637542737235034032130329934128532036135425827335528228430537929328133138529428726827325630127526127427827629028330232432929029331228128829326631427026428325628527426124026824727427527624723925223724323824823121225524224726226726423427326027425227223825124224925029325724825527526828026526030826827726529527130727329128329127327226626628327629526030925825527228725926832028427525225224623425427123725927524626925926928025627927928827526328124828023927826225126125226423924628428425225225029025230627426633630927427828726429325932337327724523424424528827226626928928927927528326228828831425127427325827729328831628333829526926725828828327328628824528924327027225925626224226526626823724126526624126024627532226926026132429427529228626231029126526826027028225825927627826727028627825028028226226728027326325325323625322021821519524423820522423120422723422921323822821323123421820618421516918520518818720219521116818317218020417521622419320122419820921017020621618518919819919315815516116215117120718618815215918715316514616616115320514814217315416016015414313716115116815015215914817516817216718314214916019312811012110414211813011811411913497141120135132136162142136150149150121146150126134130131122135128121157112 007100200300400500600700800900>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.

2 393 17500000057 510 6630001 134 867 4240000000000567 162 8790000572 681 47500001 273 169 41000002 028 088 15900005 556 848 70100510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G3.5G4G4.5G5G5.5G# 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).

100 %74 115 409100 %0 %

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.

100 %74 107 250100 %0 %

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 %8 1590 %100 %

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 %37 061 99350 %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.

96.7 %71 679 26696.7 %3.3 %

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.

15.9 %11 751 75215.9 %84.1 %

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.

11 483 547371 065171 449428 989183 667171 110513 598216 98097 791187 47581 13268 573198 03187 29148 81195 82962 97266 462111 32296 00994 93498 719100 95784 003136 164196 95230 516343 99736 34234 89155 85953 14733 95358 94333 17634 76644 45254 34125 25275 269792 09852 46758 97877 00072 913114 58395 620175 480182 29343 02346 43749 14249 52936 07957 39155 07446 149102 61249 99076 96365 850 018051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M45M50M55M60M65M# 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.

99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.99%99.98%99.99%99.99%99.99%99.99%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.01%0.02%0.01%0.01%0.01%0.01%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped