European Genome-Phenome Archive

File Quality

File InformationEGAF00008413141

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.

81 270 751155 587 509236 978 269305 833 016346 273 294352 193 629327 476 112281 759 523226 942 712172 599 613125 026 37286 842 25258 087 72537 764 76323 924 90214 944 6229 222 8225 722 4473 609 0692 341 7051 583 9781 141 507867 234691 431573 407490 063427 054374 761333 784301 900274 661249 015229 198207 791192 565179 357167 238154 576145 142136 928127 808119 401111 997103 74797 39291 82485 33882 01177 19371 94968 54463 51660 77356 28754 08150 20847 13145 68643 09941 56439 73338 55736 84435 08633 01731 71930 19028 72027 44626 91825 50824 12323 76422 48421 52021 23620 28719 94619 49618 75518 10117 60916 99717 34016 83216 68916 25315 58215 29314 94214 51713 71913 65713 61513 38812 97412 61012 21212 03412 11211 97311 43311 47811 10510 55610 48810 39810 1389 6679 8029 7679 5699 5809 2879 3259 3078 8808 4958 7478 3198 3018 2548 3267 9677 8997 7777 5977 8227 4357 2857 2547 1017 0906 9406 9786 9036 8096 9786 7046 6896 5866 4116 3886 3866 3876 2346 1455 7135 8235 7035 7065 6395 6205 5465 4705 3555 2665 1655 1204 9765 0225 1745 0394 8754 8534 7674 7674 8234 6484 7074 6194 6694 5364 4554 4364 3274 2984 3854 2694 1454 0893 9134 0394 0413 9233 9714 0653 6843 8643 7113 6043 7843 7003 5533 5243 4893 5143 3773 2483 3153 2263 2393 0853 1783 2153 1633 1643 3023 1343 0273 1203 0413 0842 9882 9822 9213 0172 9473 0112 9283 0072 9992 9452 9162 8762 8192 8262 7462 6812 6212 6852 5682 5402 5302 5902 5542 4802 5122 3872 4472 4532 2922 3462 4392 3182 2262 3292 2162 2542 2182 2252 1772 1352 2172 3202 2182 1892 2442 1402 1882 0512 2082 1232 0882 0682 1092 1062 0842 0922 0832 0122 0091 9602 0081 9461 9481 9091 8591 7741 9161 8351 8651 8301 8381 8261 8501 7571 8071 7301 7881 7821 7991 7931 7851 6871 6891 8321 7461 6431 6291 6871 6281 6661 6281 5711 6041 5641 6211 6351 6551 5881 6441 5961 6101 6891 5681 5361 5681 6051 4851 5101 4521 5111 5471 5311 4841 4131 3821 4301 4621 4701 4881 5451 4031 4301 4521 4721 4481 3211 3881 3781 4571 3541 3691 3241 3181 3231 3061 2841 2821 2831 3561 2471 2321 3121 2691 2571 2451 3291 2121 2521 2311 2601 1921 1901 1601 1771 0751 1251 1591 1281 1221 1361 1681 0941 0971 0631 0981 0651 0071 0931 1281 0551 0661 1001 0711 0219701 0141 0479891 0391 0141 0261 0551 0751 0351 0911 0891 0461 1161 0121 0151 0231 0559739709511 014962974916940912904877917898952964873876896893926889908912894895846844876910859897830848885848945789788776768782805759808769728747804716785780736725747685757695760739727771740699733792755680751753718721753725651719740720784729675720714734705704652657681732708714725679710708756731726739751700692768706658654643647679614650696661637691676670672744676683733651624663588674658595568624642641639626631657610614588643629640657627651638657622660599590579601563616554567593591633640591587584621594597599599588604558563585583535533566527567561549542558524550535522584588587614587546527543582534528516551508522513528473506533517510541544499513519516461547490501526510513500458483487488516452489439441434506463484492464479468424441485422479454469475508513464452442488455481515469473470462432457462446434486443431462433477432422423452463423420430449429447446438451427400400380399406385378403403383405407386421441421379426401361419440423432399402380437429426391401427407382390433390411382378389418406411383411410409420435411394388412389389404383357386358364390347372401345397356388356378359363374351351361311330334335357326331368344375342366337362359366398358387395362368386368394377345421459442475394382424389398373374390395382333342355354362337351328361383379359365371366364358351368396370381379385362369328339353327370339366368366356346351353381329355406360346339313350343362303334353337305334356340367341355343354350311328347321337346315342318339334322365316328348306342364341302347346360380376327338328362342331348328316304315312327331361326300372372337382363344325331345311341342349293322317290269289275256302312286259291319287290300286276299321300302297300274284292263269247282247265237242258214223229201230228204223234237212224237233219244253197210251213221216206218225195211212265 018100200300400500600700800900>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.

2 075 362000000030 854 853000748 621 646000000000433 842 4710000510 362 46700001 137 647 84100002 373 426 13700015 212 709 64300510152025303540Phred quality score0G2G4G6G8G10G12G14G# 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 %134 831 69799.6 %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 %134 482 21699.3 %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 %349 4810.3 %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 %67 713 71050 %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 %131 345 61697 %3 %

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 %15 580 81211.5 %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.

6 760 866130 23180 389158 167115 971123 816140 687192 71093 604149 05466 53158 10482 37192 57548 938111 18777 55887 788116 931167 668175 171166 546209 737157 411252 589404 28027 724686 30939 42436 98872 87779 01541 41995 61538 15438 91561 22982 31622 951125 8141 667 63384 81381 170138 906115 301213 314186 750273 138477 74752 66969 94060 76479 07737 98281 68069 13150 816187 49052 274105 393121 908 025051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M# 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.

99.74%99.72%99.73%99.74%99.73%99.73%99.74%99.74%99.73%99.73%99.74%99.73%99.73%99.73%99.72%99.76%99.74%99.73%99.76%99.73%99.73%99.74%99.82%99.71%0.26%0.28%0.27%0.26%0.27%0.27%0.26%0.26%0.27%0.27%0.26%0.27%0.27%0.27%0.28%0.24%0.26%0.27%0.24%0.27%0.27%0.26%0.18%0.29%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped