European Genome-Phenome Archive

File Quality

File InformationEGAF00002386077

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.

32 798 44778 294 838148 967 316231 397 216304 609 307349 948 121358 222 178332 878 132284 489 145226 265 819168 906 862119 396 61080 561 74552 269 76632 864 43920 165 76412 214 7597 414 7204 568 1192 913 2421 958 1711 390 8501 044 951831 750683 193574 698505 798445 712393 565351 437317 157287 786259 723240 153217 033200 131182 701172 571160 683150 127139 872130 084120 846113 576107 06999 18492 84386 11280 05775 93170 98066 22761 67857 82054 41351 35348 43546 72544 97042 98140 05538 38237 32835 41934 11233 45731 84130 66429 40028 39027 56727 09426 44025 91325 41024 29623 96823 49522 34321 83521 42820 39119 90219 96819 41419 08018 59918 66318 44518 00617 65117 73216 98016 77016 63815 65615 33614 58114 60614 36314 16113 97713 91213 69813 67913 06812 85512 83412 16711 74311 43411 26011 18111 11810 75310 27210 29710 30110 2129 8419 7489 5409 4799 2458 9409 2539 0808 7038 8668 5568 7248 2498 2218 0998 0497 7907 8507 6967 5727 6577 5297 4287 4127 2387 1096 8777 1786 6886 8186 7846 5716 8146 5546 4536 5916 6046 4626 2666 3815 8845 9525 9225 8925 5195 5335 6315 3645 5995 4985 2365 2345 1045 0224 8964 7524 8244 8104 7434 8874 7894 6594 7014 7524 7844 7284 6924 5314 4844 3504 3714 3404 3424 3124 3554 2344 2194 1944 1713 8653 9473 8363 8053 7383 7913 7573 6553 5363 6013 5333 5613 4423 2523 3183 3023 3893 3143 3873 3103 3123 3013 1642 9753 0473 2883 4023 2133 2083 2283 0762 9323 0003 1322 9372 9932 8772 9212 7592 7182 7672 7622 7362 7382 6772 6922 7202 6452 5682 4992 4682 4742 6602 4912 5482 4562 4092 4942 3822 4412 5492 4642 4542 4212 3882 3672 4372 2642 2522 3362 2002 3002 2692 3222 2442 2102 1972 2052 1842 3152 2622 1462 1172 1212 1032 0922 1072 0292 0942 0612 1312 0622 0322 0482 0642 0112 1122 0271 9592 0112 0371 8772 0101 8621 8841 8521 8241 9091 8921 8171 8791 8091 7321 7331 8001 7361 8601 8101 6661 6911 6291 6741 5621 6761 6601 8181 7201 6051 6481 6431 6251 6061 6321 4761 5731 5141 5861 5231 4851 5461 5161 4911 4181 4771 4921 4151 5071 4651 5581 4991 5001 4991 5281 4881 4821 4721 4761 4511 5901 4821 4681 4311 4791 4381 5121 3411 4061 3421 3851 4041 3061 2891 3451 3951 2991 2971 3271 2921 3051 3321 3451 2571 2931 2051 2471 2921 2041 2261 2001 2711 2611 2621 2051 1401 1481 2271 2141 1651 1271 0911 1711 1461 0631 0611 1041 0891 1421 1441 1941 1361 1151 1931 1201 0881 1191 0681 0641 0721 0871 1791 1551 1041 0511 0371 0411 0511 0221 0791 0579971 0191 0321 0669921 0021 074997978964948936971899940905999966881894894919939872922959838893836908923901894955963913932878902875909857826848817786822816815855885899891801800819790829828834873826831835830791809765833757796837798829862818825824810764829806799822830804729811776738745750800760776776842736723707703751678707685693705722656697676763697681652682645710672655684668689717699672688645654644601637651637656662683689632642683605632621629677587609607610577630609569639628635596606629619616596618550622564569584570611559648660627608585605552569576565568555560553576551501533521513516513511505500504536550535497465506487559520495501524560556540512509538504546530501466469515511488510500511543487510476473505436497475492570525486452481484516533430436476485488493474517467516523542564547515501505491528557520559544552532547498492477498440490519452478480472465487463466422461514473446470451443491485464427445462476471463490463432444486453463526505500484470492542495483523487496447447466474450452448438436453452418429466457421448466454448410429409424459424441412438447412408425450440415434479417433432391414431391356386389410381402371378397343395347346308385350396374343322349370299319312395330350349357334334374305314341321310318291334346305285315318347304309333322311319329285312321327330283297313311297263277282283271283248263276293262267279278262287248268242243289228242250264264265279261248253245246269218217275276246257233243248262253223248235233238262249246236237259234251255222259247213227247214246256233225222238233230262221241243222256216201238238249214234226229228261219250238233231221225234247223238245230200238236240264244229238229211217225213219229210216203219254182224206233221225222230239227209255232252220224212266 424100200300400500600700800900>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.

3 878 233000000037 443 7400001 043 490 631000000000575 614 2640000592 792 96800001 311 711 94500002 729 859 62500016 939 671 30800510152025303540Phred quality score0G2G4G6G8G10G12G14G16G# 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).

99.5 %153 051 17999.5 %0.5 %

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.

99.1 %152 471 06899.1 %0.9 %

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%).

0.4 %580 1110.4 %99.6 %

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 %76 935 30750 %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.

97.6 %150 185 51697.6 %2.4 %

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.

10.3 %15 809 59810.3 %89.7 %

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.

6 818 011169 680103 223197 360149 321155 850185 136238 440105 286180 10882 83071 464107 460111 96859 998133 13490 08196 816140 649179 776186 936181 121206 742159 373260 639437 48030 950767 50346 42843 70695 92490 03939 683104 66943 80343 43976 72495 77225 061141 9572 172 70595 67794 581148 383126 993231 192207 584278 470495 11959 30177 90067 59989 09246 70978 71181 13260 175210 55463 328119 143137 153 490051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.6%99.59%99.61%99.62%99.61%99.61%99.61%99.61%99.6%99.6%99.6%99.61%99.61%99.6%99.59%99.64%99.61%99.6%99.64%99.58%99.61%99.62%99.52%99.46%0.4%0.41%0.39%0.38%0.39%0.39%0.39%0.39%0.4%0.4%0.4%0.39%0.39%0.4%0.41%0.36%0.39%0.4%0.36%0.42%0.39%0.38%0.48%0.54%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped