European Genome-Phenome Archive

File Quality

File InformationEGAF00001307689

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.

229 600 593370 838 102451 802 038454 611 813396 577 907309 838 368221 544 148147 699 97892 895 33155 941 72732 611 96118 601 58510 483 2005 961 4863 478 5292 091 4131 336 748909 328654 471493 515388 770321 729267 927228 374194 981170 030149 041130 630114 263103 24390 92079 89672 32764 88759 26354 80950 18847 10943 99940 10837 38734 31532 56630 91329 31626 70825 54424 44523 28122 36721 47420 65919 46319 04917 94317 16616 30715 88315 29414 66114 42213 74313 16612 75912 42012 41411 56011 64511 27310 6199 8649 7149 3549 4979 0068 8128 8738 4758 0077 9437 9977 5947 4327 5617 1557 1587 0016 4426 4256 1486 1215 7435 5375 9355 7245 6195 3465 3425 2155 1014 8884 8634 8194 5874 6464 7694 6224 6114 5884 4614 2854 4754 3674 1203 9984 0943 9793 9063 7363 6983 6653 6433 5733 7123 5863 5833 4973 3933 5253 4603 4593 1122 9503 0843 0123 0483 1733 0923 1152 9962 9853 1613 0012 9022 7792 9482 9592 9312 8852 8112 6972 7592 5002 5822 6282 6442 6692 8272 6862 6812 6312 6112 6172 6342 5292 5052 4362 4512 3442 2522 1432 2332 3782 4142 4042 2792 3482 3822 3292 2812 1912 2592 1472 1292 2042 2222 1072 1011 8901 9872 0622 1172 0531 9222 0112 1162 0461 9451 9612 0312 0051 9801 9971 8461 8511 7721 8371 8171 8101 8961 8501 7871 7541 6621 7071 6851 7191 6231 6901 6221 6251 6961 6281 6831 5891 6061 5821 5781 6751 6021 6051 6311 5971 5501 4811 5301 5101 5771 5191 5061 3951 4171 3901 4461 4411 4171 4371 4561 4641 3991 3821 3951 3871 4241 4101 4751 3441 3771 3611 2331 2761 3151 3581 4001 3231 2411 3241 3111 3001 2871 2781 2021 1851 1081 0641 0771 0861 1411 1141 0591 0641 1181 0751 0641 0289911 0631 0389999499631 0051 0141 0249569419199449269059388688218488638278538608459349018118478288458848058138068027707277277268018207526687437807787267897658017466817276807296797346636676667126466486886496686727076376576305976116295786016296026626086405986186356506146486275756135945675365175966085625845335975465815565565895955675885826276065755055315185295565315195084684854995094845164884905485094585114705354985344724905234774984455145254644865244924695645314564884984964835184795405305375664824755074854654524685084994895295205204874874945264965015114864945105395195285085395145495065175374835174815145765405766346265696096245716145725465465575615495365685605436195495555585655505175215575155275315195215184864704574334864805174894624934834754695114944774984774734574334574474354974994964294524314334434374314514614784594054044234253953933553993803773933853913823433213703873503693683363343203203493203103443172993173262932943103122762813033012602602642632782702702322852512312722262392162512482132522032612362432302322092102742472472622292632232722162432202142482022432292192512072412242122282082281992252102362182262302302172182502162092112262291942141902002051942002041952041792102092122021972102042122271911872302011821991961932011802172101911781972141832151892001641661781961722141861872081911921781892062002021851351931902012011832172001992322112141741831971791821801841711871992081711931841681631611571531651641611721441711791771641751701561731491881461781531821721591671511651791851651751831981651631591431771651731771741691781771681881661801661621841741691711621921361581541671661741471461441491461731531731601121491761571611581621671561521551591781491581631541881681711771731591451361631551501481381521261261531581581491421431501441321511281391571461351311311261341441311491291441201381401241231121361411471111211101331201251321221181271361441191291351161301391511381241551281511161321241081171191311221231201271271131031331301151381341051081359511913112311610411210910812599102127134116118112112124131103113126123117132921251041161219512410392106105115115100129108109109111108132931051189285130123111113112112104115106114126115115116 053100200300400500600700800900>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.

1 434 51900000041 094 346000554 677 3180000000000291 286 3670000341 328 8280000895 177 01000001 655 679 15500009 816 456 30100510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G# 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).

99.9 %89 981 13299.9 %0.1 %

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.

99.9 %89 929 46299.9 %0.1 %

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 %51 6700.1 %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 %45 023 62250 %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.

97.9 %88 150 50497.9 %2.1 %

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.

11.5 %10 389 06211.5 %88.5 %

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.

5 927 167166 50578 846221 00996 822100 459285 642141 26365 600123 20349 70143 093110 81359 88532 89775 20649 24552 54779 30181 09686 09189 40695 13473 906124 197201 76422 067400 35026 81825 85250 03544 25527 31453 19325 02724 66636 80847 96717 56473 587958 62152 43250 75281 00467 095118 908110 001159 279291 67631 80945 01637 91452 11725 05443 61744 16032 932119 53234 26467 84681 794 574051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M# 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.

99.94%99.94%99.95%99.95%99.95%99.95%99.95%99.95%99.94%99.94%99.94%99.94%99.95%99.94%99.94%99.93%99.92%99.95%99.9%99.93%99.95%99.92%99.96%99.97%0.06%0.06%0.05%0.05%0.05%0.05%0.05%0.05%0.06%0.06%0.06%0.06%0.05%0.06%0.06%0.07%0.08%0.05%0.1%0.07%0.05%0.08%0.04%0.03%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped