European Genome-Phenome Archive

File Quality

File InformationEGAF00002055900

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.

42 900 80011 908 2951 651 412989 029554 508411 345315 438258 095215 853188 810167 024149 025133 672121 859109 812103 05795 78289 79083 64680 12474 11970 44567 08163 71061 47658 35856 11853 52451 57949 35348 03646 31744 72343 40542 07440 58340 04539 05937 98037 01936 18534 71934 17633 39132 33831 97231 56931 17730 16429 79529 06628 36727 76827 53626 80126 61226 36625 88525 87725 25324 93324 75724 13123 84224 01923 30422 93622 95422 68822 60922 00122 17821 90321 41821 49721 13121 10721 08520 55220 83620 36220 50120 21619 70919 73119 89419 73419 79019 23819 36819 21519 25318 90819 23918 95518 75818 46418 42318 41318 43218 19918 20517 89017 93918 24718 39817 59617 81817 71117 58117 35417 51717 61417 25117 10917 11516 88516 88016 69316 63416 67816 46916 45316 48816 34016 30416 07915 94015 83015 98415 60915 43715 32515 28315 10915 04615 12314 87314 70714 46314 47414 36714 35614 11413 96613 67713 60713 59913 43713 08213 12913 03212 79112 73212 68512 43812 41511 86812 01911 79611 60911 39211 42211 25910 91411 05810 94310 88210 60410 72110 69010 48610 52810 22710 1949 8639 9839 6339 5069 3819 4479 1158 8438 8108 6548 5498 4628 3338 3358 1668 1498 0627 8557 9887 8217 5827 4837 4317 4347 1947 2547 1817 1647 0816 9866 7366 7396 7116 6246 3546 2986 2296 1286 1966 0545 9305 7485 7665 6075 5655 6795 5955 4625 3975 2585 1165 0864 9774 9894 8824 8384 7344 7644 6324 6234 5734 3474 4234 3624 3034 2394 1564 1624 0304 0753 9453 9453 8683 8453 7393 7473 6683 6433 5073 5503 4273 3933 3883 4473 4623 3103 2293 2063 2243 1633 1183 0572 9452 9882 9602 8982 9662 8682 8382 8452 7792 7122 6252 6772 6162 6042 5792 5682 5262 6702 5732 5652 5502 4992 5252 3592 3402 4382 2832 3112 3002 2732 3022 2782 1452 1552 0972 0732 0192 0381 9622 0281 9921 9651 9231 9011 9161 8301 7661 7571 7771 7461 6951 6591 6531 7061 6471 5971 5571 5521 5421 5171 5391 4321 4011 5141 4291 4601 3581 3341 3741 3891 3801 3531 3251 3761 2621 2681 3521 2571 2411 2101 2551 2321 2401 1971 2091 1561 1491 0891 0921 0391 0771 0381 0801 0061 0801 0021 03195596894491393590794892084689795493287384880880382282674883080083983881677075976572476874475475075469973170673670469165162261765665461061966460760263057057359261060967558860056758759456358054550455154651954748948845849747845849545445250744140846043640139739139238136437234639236133731730433128933827928831926726425424325525324426226026025126624724529026426023324521322218821321822821324423321722724921720821122517122119418218218518620016317817015417315616213614415413914613913113712113811610612296998286831131191131001241091101151088711691849110398838894101118109871041181161291299110712799112114108106111103103116931001128395787986818469859298898885748674877177979165827987979667929284899711191939811490901001028495931161181421041241321291191131051231241231261101061261331251171111171211021121281121081081181111201131011121061088494738879819287818673961029289857672757278656781778910779968181100971091001061118311193106777970748870686664625945585161746351535749516440443939475339394438384545445044534862485749423647424338384435402944353039303739333841343233262928394435372934312525273723253126212119321926271828251820272620273125211732244028342437232623242029162033172023141691713111919182321232220191130202319172226151817111623121717101515191815161313122518161412181618241823152417191616242025251313201218172117191321131925242313192118251314152315231719202019181114229181616121513713222112111114209111489789714915131417111413131410810131589812826161014151015131038916109671014101114141311155133 319100200300400500600700800900>1000Coverage value101001k10k100k1M10M# 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.

02 32614 018 13836 09955 674189 198215 268342 640391 021536 228583 393536 177431 658323 209341 007459 606582 224722 2501 024 9361 192 229925 876826 620661 0701 309 5832 556 4983 697 4253 803 0045 256 2038 112 14611 321 77933 413 78550 782 54275 602 179112 876 242103 596 16668 495 03647 633 71224 804 26613 737 61011 299 449000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M110M# 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 %5 952 90099.8 %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.

99.6 %5 943 90099.6 %0.4 %

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 %9 0000.2 %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 %2 983 63650 %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.

99.1 %5 910 69499.1 %0.9 %

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.

5.5 %325 3825.5 %94.5 %

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.

161 1399294712 1805034229621 2506973 5101 6544028718853632 4222873498911 1772 9463 2941 2209594 2877 25716028 2642861941 0898571752 2562272177528933322 94530 5886711 7331 5411 6384 1101 9245 4106 94710 31629 5211 5822 5569531 4663 0434644 8566808745 618 6572111111 8870510152025303540455055606570Phred quality score0.5M1M1.5M2M2.5M3M3.5M4M4.5M5M5.5M# 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.

99.86%99.87%99.87%99.87%99.87%99.88%99.86%99.67%99.86%99.86%99.86%99.86%99.88%99.84%99.87%99.84%99.84%99.87%99.83%99.87%99.86%99.86%99.85%99.85%0.14%0.13%0.13%0.13%0.13%0.12%0.14%0.33%0.14%0.14%0.14%0.14%0.12%0.16%0.13%0.16%0.16%0.13%0.17%0.13%0.14%0.14%0.15%0.15%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped