European Genome-Phenome Archive

File Quality

File InformationEGAF00002339634

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.

834 821 472698 843 497429 267 215213 762 84791 632 02535 268 55612 685 3404 537 6621 751 263828 231474 497329 878249 717198 124161 096131 850111 27498 53687 18578 04269 91463 35658 42254 08747 82546 34542 29039 89636 93036 22234 35532 68331 80328 70527 94326 04424 70523 42722 70921 53220 71119 86119 03417 93617 42516 85016 45415 50015 61314 48614 27413 60612 65712 25111 70611 25111 25710 37310 3399 9489 7309 4219 0498 9278 4268 1877 8828 0657 4707 3397 4137 2836 6756 5556 4646 3906 2066 1526 1185 9805 8435 6255 7125 6335 4825 5325 5245 1035 1145 0434 8885 1074 9334 7924 5914 5674 5284 4274 0313 9164 0413 7683 9543 8893 8373 8643 9593 7513 5473 5063 5843 4693 4803 3943 4383 4263 2703 1332 9872 9463 1652 8672 9242 7272 6902 7502 8742 8202 7482 6582 4992 7942 5582 4822 4882 5322 4402 3312 2722 3332 2552 0502 2342 2662 1042 0432 0272 0191 9171 9051 9121 8891 9172 0501 9532 1311 9491 9271 8691 8201 7771 7981 8021 7761 9371 7881 8061 6681 6721 6531 6591 6251 6001 5121 5871 5201 5861 6181 6061 5791 6181 6271 5901 6521 6041 5851 5011 5401 4491 4481 4191 3111 2671 3391 2661 3171 2961 2681 2751 2801 2301 2231 2131 2331 1921 2751 2051 2121 1581 0741 1801 0761 1041 0611 0681 1441 0861 0659441 0141 0039719369398969249809228759219189189069228818888398208118398328058078007577857597737888038077437517987438427868218418427908588258898287948209118277987918408178778808878998559029348698859188948708748498988497988428248418018568658848267807577628027447647417127557327057387187527317596857126806797006536236456786956196366436276637187046767326506676896306426366596276396236386516066526566596716656606586936416486196395706236335956296206535945825556856326146155995785896366636316336696946296365946286416275796596535965685195515705164965195235025184884784514964254134754334464614324784224124164254274274545804073894124034133683903633994084203974003723844033623913533583893553803523744013503483343542993263323383293263333313373483324023744083833743643693163153253193082843033172722732792902683302802752662532742593002752612702892992993252773082682732903002922772762712953102632942702712482562623042692842792762292492512582712412462672322332572452122112582462342192241652312321851901642091921592021892381851872061792022131892191701751801731801951771751951911491791881761972101951771901601781801911541501411851641751821881451581631731931611621701751541561671901321391581541431741561681471611861531691861851882011661631851901681381711431781441641591521431591521291471351571531181461521821251491181501341481431771531591451251731401551531501421331351211381521301221301511161371301271281371111101201201091341341021191171321301099911111811412612212211312410812012013311510111912511212511712511311413215413313010713414012497118114123123129111125129135137122136155115120121135129155115138141124981341201011161211291201181211231041211009110810110912211410412511112011013311490118112109891091071039411010411795928310072891008195947588861171029692899910089987278779280907876977672707187758683106798477818881868976829282797210094788078817876797373839298727784869898951018799979186868581909710291105941079889879185758788839173116105100100919488837670958176651028090968792968275708277776976636275647166687274777260677278716281727770718269676970588560707858897168876482805777616879726874849482707187748580767071736564877470695876877976748472636862647381696335 347100200300400500600700800900>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.

883 031000000043 719 432000395 457 905000000000220 137 4560000242 404 4190000437 844 2050000812 452 5900003 321 511 73800510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G# 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).

99.6 %36 118 31599.6 %0.4 %

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.

99.3 %36 002 24299.3 %0.7 %

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%).

0.3 %116 0730.3 %99.7 %

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 %18 127 18850 %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.

98.1 %35 570 17898.1 %1.9 %

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.

5.9 %2 149 6415.9 %94.1 %

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.

1 465 88834 67620 98641 06129 92331 16837 19147 51920 08036 77616 35914 44722 21423 31111 53228 28818 26921 43729 35836 35437 12237 73244 23134 04356 90594 7645 966169 3999 0768 99520 57718 3757 54022 9049 1308 73316 54320 3644 99629 632503 90521 63422 11833 17729 20052 16945 92268 844103 34714 74018 06117 05220 32711 90418 86520 80216 11849 70017 49930 98032 611 188051015202530354045505560Phred quality score5M10M15M20M25M30M# 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.

99.67%99.66%99.67%99.67%99.68%99.67%99.67%99.67%99.66%99.66%99.66%99.67%99.68%99.67%99.67%99.69%99.68%99.67%99.67%99.67%99.67%99.68%99.78%99.52%0.33%0.34%0.33%0.33%0.32%0.33%0.33%0.33%0.34%0.34%0.34%0.33%0.32%0.33%0.33%0.31%0.32%0.33%0.33%0.33%0.33%0.32%0.22%0.48%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped