European Genome-Phenome Archive

File Quality

File InformationEGAF00005190907

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.

7 377 60512 025 39821 462 02736 441 32257 574 93084 547 450115 715 982148 502 422180 019 787206 888 607226 309 519236 076 992235 411 471224 965 791206 630 051182 660 059155 839 389128 702 996103 010 19280 182 14560 806 74745 007 33032 722 35223 363 13616 452 93611 456 3197 935 3475 476 9023 780 4232 634 9901 852 3861 325 634976 587740 391578 704465 682387 329328 297284 821252 469226 651205 682186 837171 902157 522146 830135 657126 016116 837108 529101 50295 11989 39683 17177 68172 87768 99664 94060 79557 98253 80550 93548 66946 90045 20343 41640 85940 21438 43036 92636 00935 16634 03133 27032 43132 03931 94630 26229 41628 25927 55826 71925 87325 56124 65924 08323 00221 46220 53919 41719 40118 66718 38717 66017 02116 53915 69614 86414 43713 57113 17913 33812 40012 09911 49111 43110 99910 5729 9409 5089 1499 1078 5878 7118 4538 1257 8137 6597 7347 6677 4107 1026 7186 6026 2816 3586 3386 0646 0145 8175 6305 4565 2455 2465 0244 9774 6604 4724 5674 3504 3174 2194 1113 9544 0124 0253 9494 0933 9983 7833 9654 0443 9373 8793 8373 7273 5753 4413 4133 3563 4673 3253 0663 1223 1432 9833 0243 1032 8372 7062 8422 7152 6812 6892 6962 7942 7782 8052 8602 5992 6512 5672 6742 6022 6172 6442 4492 4162 4102 4582 3972 3822 4032 2272 3262 3422 2792 2192 2022 1622 0902 0152 1231 9812 1572 0151 9771 9032 1821 9511 9462 0041 9401 9221 9221 8321 8591 8431 7111 7621 7141 6861 6881 6641 7351 7691 5781 6161 5841 6001 5321 6911 7021 5861 6981 6221 5881 5501 5371 3991 4091 3131 3931 3811 3141 3811 4461 3901 3771 3891 3631 3741 3621 2901 4261 3171 4011 4041 3391 3041 3341 1601 2011 1861 2211 2301 2371 2511 1591 2331 1521 1721 1581 1421 1501 1641 1461 1371 1001 1511 1821 1231 1151 1611 0361 0641 1771 0711 1061 0441 0671 0771 0931 0721 0671 0721 0571 0561 1019401 0191 0449991 0049951 0359449639219689408599029328749561 0321 009964934896908970861939878886889888939882897903824952866899931827771760769831807768792831781851764653715760677650703663698693700649651648688681705729708659636660625642695717671685679673661732706751690651645693704693651644627667671637657711660621671649656652613651632714714745759646667639725576558566587566589566558549523544452524570533509510551560558559608608553572557537545548504472568520485502494479450521522498468467503504490484495486467479468539563537532556517546495553572565495503523452498514469479479460458431466508402475444428442417400447457408488404424391422412429407390360371376432367397394392381417412377369355369369398380388338464415418415469395369385394380387399352368406379346411359376362343329367393398401329348363325302359342356330341379369319346365352300345386352350420372372330338337375339294290338305358331314364361301306346356327342332291327344308298311317329301294292285299293292285285286298268258287296310272287267274251244244259261265265253263264251281276298250263280279248243272281362300277261295256256284268281266270273245238242280263251274285268272285276262244269229267249238254246267268251260228286302242252247218242264201237220253201216234215244241261246217254212248239248246221205229229266210253239237238238235215226225222192235194212188214222196211195208219213198207187196196195198216219215205210212214218197236218230230240217222233204231211253211266223244270239241229268293274222225209201244192220239245208233206199208227201220215214215206188225229226225208190185188207203184216206225202211180186185182210171204194205195177187186221157186185194195206225233235186185185201207215212189210214218204219181206192201209208209187185219244193214192216211197189174183164206195184209176180158191182159188161184173202177168189175156177173178165166154189179186211165169205205166226180214176204199267224179171208190171202167173191198181197188187202170177151165173207176179205192189199171187182175184203159198192183181174170187166163161167181193167160179159149164141156154154165163170193160182175155153184149195160179164160170173180176 286100200300400500600700800900>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.

602 21800000000001 213 112 70600000000000001 867 947 6950000000000035 799 935 30700000510152025303540Phred quality score0G5G10G15G20G25G30G35G# 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 %257 080 84399.8 %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 %256 719 53099.7 %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 %361 3130.1 %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 %128 747 01350 %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.

98.4 %253 329 46698.4 %1.6 %

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.

3.8 %9 694 8673.8 %96.2 %

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.

5 920 371114 10269 819164 91595 177100 412119 600216 26288 330135 03456 54549 65872 33877 32548 364109 65863 07269 60589 263117 600123 283136 921158 123124 729208 319380 70424 829923 88933 69132 06688 36265 63538 00590 71735 21434 87158 51873 15224 316131 8682 515 178113 93294 127178 187141 426265 225268 403336 720814 54860 891103 18275 706123 87946 61686 19683 77159 248295 75159 491146 521241 616 141051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M180M200M220M240M# 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.

99.86%99.85%99.87%99.87%99.87%99.87%99.86%99.86%99.86%99.86%99.86%99.86%99.87%99.86%99.86%99.86%99.85%99.86%99.84%99.85%99.86%99.86%99.61%99.54%0.14%0.15%0.13%0.13%0.13%0.13%0.14%0.14%0.14%0.14%0.14%0.14%0.13%0.14%0.14%0.14%0.15%0.14%0.16%0.15%0.14%0.14%0.39%0.46%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped