European Genome-Phenome Archive

File Quality

File InformationEGAF00001349002

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.

438 375 379411 446 069355 337 850297 254 578239 206 973186 357 349140 282 371102 827 18273 653 86651 930 18336 178 58025 266 99517 814 97012 758 1659 393 5307 130 8305 577 2894 524 4053 784 3283 245 4582 836 8192 512 6732 249 6752 021 9461 836 5151 677 6791 544 2901 424 0421 315 7051 217 8531 132 6181 058 107989 133924 821867 222818 249768 861724 956680 073641 014606 378572 446544 184514 261488 168461 807436 767413 470395 113375 946354 270336 835319 516304 271292 477277 190264 824250 729239 746228 957218 150208 353199 388191 005182 541176 480167 293159 137152 098145 247138 828133 166127 170121 557116 110110 585105 585100 89696 68893 27189 85786 29782 53479 87375 67172 30869 21066 59364 06862 26159 28257 11254 81252 95350 34648 26146 18944 74642 77540 91139 42238 59337 20535 83534 28232 88932 23231 06329 77428 85827 33126 62325 76524 67023 57622 93922 27121 64320 87120 02019 23018 70318 04017 49316 48115 81015 17714 88714 24314 06713 60513 00012 53312 28011 83811 52610 98510 85410 58610 59410 2639 7879 6949 1598 8208 8038 1798 1347 7897 4667 2807 5036 7636 7296 5786 2676 2655 9846 1745 8575 7835 7045 4815 4525 3445 4365 2325 1784 8764 7384 7814 7144 4264 4014 2284 3164 1033 9733 7813 7813 5813 4943 3843 5533 4863 4263 1733 0863 0933 1173 1773 0792 7652 9642 6952 7642 7382 8262 7452 7132 4292 4992 4032 3692 2952 2682 1882 2482 0982 2432 1432 0912 1342 1781 9311 9701 8711 9151 8761 8451 8571 8711 7641 7771 8221 7711 8211 7601 7391 7591 6481 6651 7911 6101 7091 7081 6261 7091 5781 6111 6091 3931 5431 4881 5371 4731 6191 3931 4761 4881 4911 3201 4881 4681 3961 3781 3631 3921 2641 2171 3471 2951 3231 2941 2711 2521 3161 2701 2571 1751 2321 1511 1771 1611 1611 1051 0941 1531 1831 1261 0851 1591 1511 0841 1111 0621 1151 0629941 0711 0669601 0539989759859189461 0169339919929129309439139329909479611 0521 04197296889585987997190594388777488484996293985384191196486181083880879080489082681380079180082578080378079882675875480984176784578583075084585381878180777677776084177784982378784878377780876871879278088580081677276578471781572576970674880085683379177172571576976277578372272870074473770567376571867769273472867870564473371171868869169963067467065566066867073469157560763660868560762162460863464760358062661257964162164961662667565269060463567859764062358258451658456762361960555657759254957655558357458056454853051953756154554247350051545053248148749348251550749548247949150646548152147946746052049053048452047942249048845248347747048945052749046851453749748248247642439237737140337537540739838340239736837640040736740940439640038040840541142441240839538136435036440440738537336634840742540540533936734033831935834433531432634830934637232833332633231230732530733333933432836635535433734734030531432529435231738629930027230329630833030827130831835828729331426128529029028930233429129828137831229831431926323926124424027523325825625025624626025223724627123925624325127926024925526024625725524628425627227626628425722823026424622425723521523026024326124728033226227124825626325825024022119924137922227022222823323919021921823323719823925923124125422022621825726425025023723019724122120825723923722820020223623125122420619524119421022523920720921017623321220322720919723622019824125325926923824724819920619221423517725525018219718518822319818120021218517219018917619819418020316320419119118115818420317317916017720117119319419717517216916518815118216019716316220717017616720617516716619316319117516513514015118115515814818717016417217315817016613616113614515515815118416714615717814311916314615416414616817012715715117317220717016416014815215715215214713516313715913917015715613813314712614314813413215613713012512214516614113813912815414114714414413715514614412716312312812411611512810811612515712812113713712013711313113212612510712514611712213213913414817316812115111713513414311010413512514212311411411311811510612412710813310710710099123121114112118109116103107106109 740100200300400500600700800900>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.

00241 506 525002 201 3362 711 08315 982 56314 736 2098 196 14512 049 6075 180 4494 119 3498 672 3403 570 83012 076 72211 480 72514 606 68825 744 79616 359 40328 508 05112 682 83223 495 22539 686 92448 670 02261 134 47288 065 04692 073 09371 147 631129 846 425211 191 759327 302 094247 563 537421 200 591853 814 1171 864 473 750669 065 1761 144 668 208709 076 9481 256 733 6871 386 075 0062 588 153 43600510152025303540Phred quality score0G0.2G0.4G0.6G0.8G1G1.2G1.4G1.6G1.8G2G2.2G2.4G# 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 %126 463 09899.8 %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.

99.7 %126 354 06299.7 %0.3 %

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 %109 0360.1 %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 %63 369 11450 %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 %124 055 38697.9 %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.

0 %00 %100 %

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.

6 566 798165 636106 984175 620153 832117 352137 610146 23764 727147 81380 99883 078119 934123 44296 904153 631106 247100 097123 272138 22690 896154 578150 816136 994181 654259 24445 145451 25763 25556 05482 32580 65440 722114 77745 71853 81074 56194 22528 997116 0891 887 864225 428310 304178 127793 315265 467103 300171 00770 97254 02844 031199 192192 800129 7181 001 910360 781148 930304 290278 453269 439109 267 087051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M# 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.

99.91%99.91%99.92%99.92%99.92%99.92%99.92%99.92%99.92%99.91%99.92%99.92%99.92%99.92%99.92%99.91%99.9%99.92%99.91%99.91%99.88%99.92%99.81%99.91%0.09%0.09%0.08%0.08%0.08%0.08%0.08%0.08%0.08%0.09%0.08%0.08%0.08%0.08%0.08%0.09%0.1%0.08%0.09%0.09%0.12%0.08%0.19%0.09%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped