European Genome-Phenome Archive

File Quality

File InformationEGAF00004199180

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.

73 554 969155 273 805252 624 028339 572 949391 692 536396 524 145358 567 620293 324 271219 610 064152 122 49498 252 21159 750 75434 552 14419 157 59110 364 5405 579 7713 078 1271 806 9151 159 493813 226621 630508 107428 154370 310321 441282 023249 044224 177202 457180 558163 699146 899134 781122 288112 182102 42994 25984 83978 43972 40966 02460 74057 01452 20447 74844 25841 89339 13136 01035 01533 05631 33929 93027 83926 84825 77725 13823 99721 83621 60021 01820 30119 43318 63317 96717 89417 50817 12016 25016 24515 62715 28714 60414 42514 41813 81513 30713 43312 88312 54712 04812 01911 97611 44711 04010 82410 76710 60410 2299 9469 8029 4239 3768 9038 7978 5788 4558 3608 1298 3188 0307 7947 7137 7807 5717 2497 0687 2737 2507 1516 7056 6646 5616 3476 2346 4336 0146 2066 1906 0445 9405 6955 3485 3485 5315 5065 2505 1754 9144 8364 6854 6124 7294 6934 5244 5334 6204 7874 3904 6124 4224 5284 4514 4074 4124 2654 1334 1954 0333 9913 7773 9033 7963 7153 6653 6503 4973 4163 3343 2683 1383 1443 0453 1473 0763 0923 0933 1203 1523 0492 9533 0242 8182 8112 8352 7792 6262 4952 5172 6222 5522 4972 4082 5462 4412 3292 4512 3572 3152 1752 2062 2392 1422 0942 1392 0862 1452 1382 0001 9831 9762 0511 9632 0111 9641 9792 0131 9901 9431 9101 9201 8761 8921 8891 7421 7771 6891 7531 7091 6631 5311 6491 7411 7211 6081 5911 5531 6121 4791 5871 5351 4651 5421 5761 6091 5251 5151 5391 5461 5771 6561 5391 4291 4561 4321 5151 3921 4511 3991 3601 4521 3291 3111 3891 3901 3771 3311 3531 3931 2451 2561 0941 1921 2061 1951 2051 2171 1251 1921 2381 2181 2061 2091 2021 1981 2091 2041 1721 2301 1311 1671 1841 0891 0561 1401 1201 0571 1001 0811 1061 012966997926921963920925972987983934949925946944918939936878958873917866806828849844852820859855891864906866782815749815797760813751763732709714724706696672708714675732744697676647626635620613628630609587611590582560633635766687589616587553561635575528594563536493531556492544541580571580581581563560567627622535523603533520489500506523506516481543515505488499427464495489492450462483506572528514532461484473520476406426440461416456503504443442444450485510501471511501450456490487511475492471447441438430457460463467461424453462436451479502478456455416459455438440440478465440419488431402402439442430396419400435432427451413391418446395406401410393388413476384388415429449433411415391401360369445419394442430380385388424420401419386428392393373380392378381352391421373369339358354296356341379368365369337361376370387355404358404447352383376351339375362383328353363367358431372328298347332361379354358384369346366364335360335371336369377336311369315317330329325343315297309314301291372328329302303272338346342323308274304297321338322342351341325322324303307281305338319310302308295311327335298323298300276274290292285298314312283285288306245275280259268267253267209224275239255242250215238263252234249243213247246221240219216235193212215223192209238221222232214232213197231223246205204199226195200194206216224192207213230225176233190226226197198216219228229191185198221198224217203195199194198228194218188171215178214201190200181192215220226196189200214208193224214222207196172179186198168214155190185185188193194209186193165174194160202161195194176192169185188179176181180190184180191155170202156156188159172185169164159164185164162195128150167157167183167160153169155172170173180172157185174174180181169174194170181170181188194191202307182197194200157189180213166182173198171159170166161198192153159161174155180186175174170138161174162158156177163159161166184167154211186174146219170188172144204164167163173167159160173193138182179164172162179189155170179153173187162137164159186175136160153169162170153149151174159162165154134139156155145147155147136157160146155129140161163151144171156132153139164141140135135154145146175168141116 526100200300400500600700800900>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.

1 549 545000000054 014 6780001 119 273 558000000000607 257 8030000654 677 04400001 330 312 21300002 718 995 53600012 246 706 61500510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G12G# 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 %123 559 61399.6 %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.

99.2 %123 097 02699.2 %0.8 %

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 %462 5870.4 %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 %62 029 09650 %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.

98.1 %121 715 52698.1 %1.9 %

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.

4.3 %5 276 0134.3 %95.7 %

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.

4 051 84277 67747 81597 33265 40568 54189 751106 01450 62988 66039 26734 11256 46854 16028 75670 35444 95348 44068 25577 94878 84088 81096 51181 736143 771240 51016 229503 10723 09022 05655 93544 24021 59858 65222 87122 19743 28747 88815 03680 2461 445 54966 07467 446101 75588 745158 194145 659213 527351 01844 35058 31052 07768 63138 23264 32462 84448 757156 76753 80996 449114 054 748051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M# 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.

99.62%99.61%99.61%99.62%99.62%99.62%99.62%99.62%99.61%99.61%99.62%99.62%99.62%99.62%99.62%99.66%99.64%99.61%99.67%99.61%99.62%99.62%99.69%99.57%0.38%0.39%0.39%0.38%0.38%0.38%0.38%0.38%0.39%0.39%0.38%0.38%0.38%0.38%0.38%0.34%0.36%0.39%0.33%0.39%0.38%0.38%0.31%0.43%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped