European Genome-Phenome Archive

File Quality

File InformationEGAF00004840998

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.

93 636 630164 317 945231 740 719281 968 809307 597 910308 270 417288 955 690256 280 310217 378 711177 683 278140 635 101108 225 54481 168 25259 513 99842 744 33230 168 22720 901 22414 293 7209 645 9916 459 9824 309 2172 885 9741 944 1291 324 795927 914669 636500 852389 058314 327263 693225 706198 335176 289159 075144 805134 382125 164114 299107 098100 09894 09187 47382 81178 33873 97270 08366 54962 43758 19056 24152 66648 63845 67543 48041 05539 61237 67435 08233 70332 03430 35429 02128 32126 83725 06024 84423 96422 55721 53820 43519 25619 20218 86618 38818 03417 67517 10216 67716 29116 30815 76515 26814 85214 14813 68513 61213 31213 01712 70312 51511 85411 65211 39510 99610 74411 01110 65010 06810 1939 8859 6369 4959 4059 2319 0238 6478 5228 4398 2308 1578 0008 2517 9347 9857 5087 3927 6837 5367 4317 3037 2547 1966 8966 9866 8966 9566 4406 7846 5696 5046 2106 0495 8735 7535 6805 5755 4595 4325 3565 2835 5345 0865 1414 8544 8534 8274 7564 6294 4584 4054 2864 4984 3514 3664 3504 2214 2884 2794 2304 1344 2554 1644 0853 8893 7133 8123 7993 6723 5203 5153 4053 3933 4163 2853 2543 2593 2503 3593 1423 2143 1433 1193 1273 1293 1063 0602 9632 9732 9582 9743 0563 1712 9642 9432 8692 8482 8932 9322 7032 7182 6232 6662 7042 6682 5742 4492 6352 3362 4692 4142 4212 4502 3602 3692 3432 4132 4322 4502 4252 5842 4012 3852 2872 3122 2782 2312 2212 1642 1342 1692 1022 1122 0352 0642 2582 1212 2162 0852 1172 0621 9311 9161 9902 0031 9301 8741 9271 8731 9301 8451 9331 8041 8091 8611 8711 7951 7451 8811 7701 7811 6781 7821 7531 6631 5841 6841 5441 5961 6361 5841 5251 6901 5801 5321 5051 4941 4861 5201 4811 5161 3871 3031 3531 3741 3681 4571 4241 4001 3851 3261 3281 3221 3231 2841 2431 3171 1741 3001 2581 2951 3131 3261 2291 1971 2481 2341 1861 2301 2001 2351 1751 1091 1521 2271 2091 2161 1911 1711 1351 0961 1191 1351 1591 1281 1351 0911 0781 0971 1561 0651 1101 1851 1219791 0441 0351 0171 0211 0121 0671 0031 0599499861 062983972930949906993938865864879907886887875825893829920920884853890885858856878859842859886845831891860869914899870824879823806862854935840808817845822829820811791803752779771725718743762666682634690667699656642676645697717680677692681771714673651663632600650629609628636622633645625637628620598626583592611641669616640608627635624616592562588579566616609590569581571555576565586574585543589574547596573567535559542498530499508547470474482485473461502472494509526514483530507532524467511457449471470444476458425433477442455496489470466416451465436447444460473435411439417425435407474424455456471415441442441431448422393461407405443456468509435415422432397387436384433416437400416439450431405375389395383421407426397468406422474400426405400361339350371354349373357330340333367341334347334373362369378366353320365356329342362341332361382311334344322323354332313310294358349267339356338331344374307335315346328317316305301338297333324311316318289317309309281306328303326281314297304304294299274331329284324302296321318286288286330316305317281294296285270290268289271271265330291326300312305284291278327264300282293297271314324291331331340315317299334313325345316301295281319316340320300332285307326260325295321300285325305285287274281305283300308303295300283297290275310287294278307308316272326304287319292281304286279327307341285325289329323317281302278314322313292312274296318305352318320320282268322323273275309291287301296308286306301296295281282293299272285307274266285260283304265306275255307250289281294292276255305284280262283246260282269297262253269271274279299305301307326322280306310305302302318316285330329311294309318308312305321291269318265286271278292294297314294280287288315271288317290296237259274266260277253256272243247273279262237256261258256253237223247251264258272220225245198206216210234215203225250213213237247236278218241235215247242206256235226266215248228241239210255243224240251229226243220235226242226246243224231213217184252212209211205192199 369100200300400500600700800900>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.

18 855 588000000036 690 662000752 629 214000000000446 425 5050000511 210 18900001 180 038 89700002 568 172 95100015 827 768 86400510152025303540Phred 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).

99.5 %140 639 13399.5 %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.

99.4 %140 453 94899.4 %0.6 %

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 %185 1850.1 %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 %70 668 18550 %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 %137 933 48697.6 %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 %14 577 79210.3 %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.

5 593 63796 79460 843119 35985 68990 445102 190137 41169 879110 19750 04144 83362 09569 11136 86985 07458 47167 08686 856115 277119 371122 115148 725114 370186 561307 93620 680588 79528 12227 51258 94556 50130 08272 27730 07429 67947 28864 26819 03698 5411 441 76570 34865 459112 84392 131172 498162 174224 340416 60445 12459 46951 93769 880256 32872 04861 93744 108164 70644 10389 934129 520 737051015202530354045505560Phred 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.

99.87%99.86%99.87%99.87%99.87%99.87%99.87%99.87%99.87%99.86%99.87%99.87%99.86%99.87%99.87%99.87%99.87%99.86%99.88%99.86%99.87%99.87%99.89%99.86%0.13%0.14%0.13%0.13%0.13%0.13%0.13%0.13%0.13%0.14%0.13%0.13%0.14%0.13%0.13%0.13%0.13%0.14%0.12%0.14%0.13%0.13%0.11%0.14%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped