European Genome-Phenome Archive

File Quality

File InformationEGAF00003441170

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.

157 762 777269 484 365353 498 230388 539 860375 593 676329 125 470267 149 815204 713 465149 799 022105 931 63773 122 23149 782 15933 589 45922 663 96815 318 78010 416 3967 161 0904 950 0583 473 9182 466 8691 788 1081 313 273981 444757 453595 064482 699398 527335 445289 895252 825224 618202 170181 016165 856150 796138 984127 355115 010105 85396 91392 02584 45277 73472 07468 77764 06759 39656 59653 71751 20948 71045 80344 59943 24341 64238 99737 74836 35334 29033 31931 90130 41629 26628 21527 16126 96726 76526 22624 89924 06023 12422 42822 05421 26821 06020 11819 71619 07719 34318 27917 86717 34516 87816 43815 98116 02115 88015 49014 88614 60914 70313 68614 04513 50713 40613 25912 95512 64712 46712 50512 09911 65311 45711 15511 04310 82710 56010 28310 35110 1289 9409 7059 7709 5849 5129 6759 4069 2399 0238 9128 5088 4688 2438 2978 2258 0978 1367 9497 9368 0427 5557 3967 2677 1977 2876 8157 0036 9486 9997 0626 6946 6926 2746 4046 2616 2406 0706 0325 9505 8605 7375 6265 7175 4925 3835 3395 2815 4585 2915 1285 1055 1765 0764 8824 8504 7024 7984 6634 6184 6574 3894 3154 6144 3374 5034 3224 0914 2294 2594 0024 0223 9363 9743 8783 9213 7693 6923 8503 7033 6633 7093 7473 6493 5803 5273 3883 4933 4533 3133 4683 5093 2833 2783 2083 2793 1923 3283 2443 3023 1563 0903 0993 1833 1153 0653 0753 0442 9483 0592 9372 8162 8862 7352 7542 9002 7872 9332 7582 8382 8902 6662 6992 6252 6642 6442 6462 6872 6142 4402 5522 4332 4542 4632 4872 3742 3422 3752 3222 2232 3462 2482 3012 1932 2232 3232 2692 2042 3042 2142 2242 1622 0602 0732 1622 1682 0872 0372 0522 0092 1242 0902 0581 9861 9361 9851 9991 9561 9712 0241 9541 9461 9921 9331 9591 9671 9861 9621 8341 7651 8021 7151 8331 7511 7241 7451 6031 7061 5851 6451 6381 6381 6201 6231 6341 5831 5251 5501 5541 5711 5071 5241 5941 5371 5351 4781 4351 4581 3831 3741 3851 4591 4931 4291 4871 3621 4411 3631 3611 3741 4091 4521 4011 5151 4421 3841 3061 3101 2181 2161 2521 2381 2641 1941 1891 1401 1621 1381 1601 1741 1241 2141 1231 1531 2171 0911 0991 1321 1381 1221 1631 1221 1711 1651 1081 0911 0401 0951 0121 0071 0591 0069891 0731 0371 0221 0139981 0611 0169731 0081 0159759859711 0489701 079932983974966976956968976939921935903867907917946866908919980940927931892926875906879915939904923992872876903869921870820835779799840840847763724756801765815795798732796790799774790763776799812808849797869801809769777787779754750792777756830820723803733768784706776719733698714733728707730704672707662713778702684664632720697639674641637597643648646629644627671647689586589632621607604630626651678617584619576591608625560551542550512550540554518543559537538566567514563537530564558535565480547538543551519551515499497535505566534526484566586498505558445537509521495500522479538465491518506479495476473493535509478513466463470467474490473482470470523491511480454462507449458459546570503475503481449492503453427464483471475500526422494455468445434457427411447461434464448472461455409457453424455437438458441428385422417432445451414433457466434433458462440465425444432409425415432411447415439441415461428427449452389393423395414484467403407398402412388450443431446444443417468408454471459461458517439508501460462481436431434436471461462457461479496480483445458503500413457445443499479487478487451457459491500463434441472447466463482456483509468469457492517480415496435446485422422394427424412418389447407411428410421424407437403410362444401433415471436411403434417402432414407424425415445391395404425424420401391414424407422406435444431427452389381400379393384366395388387373428394412429410419397405373365405345382396404376392401363369380398361391400403350379377384360372308351354370338328346319345346343385367352378342336317354325353353301318336371348315357321324301306301298313300298313276304263310302287274304298278307304319284317288313319292286298276290252279281279256274260246233262263260241274253272255231251244228262276266228248243250227264250259240249252248234292250223243234188237223206231219212227203205165215193188211199219178218232 871100200300400500600700800900>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.

2 148 600000000035 260 160000889 536 167000000000501 647 9490000562 202 26200001 176 297 37600002 435 519 49300011 659 418 67700510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G# 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).

99.3 %113 536 07599.3 %0.7 %

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 %113 296 05699.1 %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%).

0.2 %240 0190.2 %99.8 %

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 %57 159 04250 %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 %111 570 87297.6 %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.

6.2 %7 141 7526.2 %93.8 %

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.

6 034 429121 15476 781140 099103 799107 227123 577159 81174 548122 76759 62950 42473 17280 48145 67095 53673 00378 170101 619134 288151 819134 609161 110117 514188 606314 75522 484568 61933 53930 99064 48163 15228 90675 97431 72131 04152 06566 22718 462101 4501 504 49068 64067 681109 42291 947172 999148 934226 558359 13144 28457 59350 59364 55433 15852 66059 71344 552151 50747 02188 851101 436 242051015202530354045505560Phred 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.

99.78%99.77%99.78%99.78%99.78%99.78%99.78%99.78%99.78%99.78%99.78%99.78%99.77%99.78%99.78%99.8%99.79%99.77%99.81%99.79%99.79%99.78%99.86%99.79%0.22%0.23%0.22%0.22%0.22%0.22%0.22%0.22%0.22%0.22%0.22%0.22%0.23%0.22%0.22%0.2%0.21%0.23%0.19%0.21%0.21%0.22%0.14%0.21%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped