European Genome-Phenome Archive

File Quality

File InformationEGAF00002872535

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.

51 810 653105 195 926170 835 318235 836 102287 073 940315 536 195318 084 910297 915 854262 037 664218 187 756172 955 323131 532 84396 302 87968 216 93046 953 28831 571 88520 826 25613 573 9288 769 0235 684 8713 716 2522 484 2821 708 2841 223 264919 099712 783588 444493 907431 000379 741337 448303 678275 841253 328229 302208 639191 761174 880160 481147 616136 120126 674117 563107 134100 22392 44985 98181 03375 93171 40766 08563 29258 82656 32253 71151 61248 38745 59643 54642 66740 54138 84137 58736 24134 96733 22632 83531 84330 72730 24129 60828 12327 35626 74725 79224 69723 70122 74722 31021 93321 39920 71819 82119 51618 64318 09017 35717 27016 79116 23216 03915 17814 98214 80314 30913 81713 75313 69612 89512 90012 57612 42311 92011 76211 28611 22011 15011 35911 04110 77910 50510 4659 89710 10910 1789 6399 6499 4499 6379 2309 0388 9568 8528 6428 6348 4768 3558 3978 2598 1587 9687 9837 7167 5957 4217 3587 2097 2967 1697 3327 2117 1307 0426 7656 8386 8406 7526 5546 2696 4986 2336 2286 1336 0615 8165 6285 6415 5915 5465 9235 5255 4915 3385 3485 3755 1355 1785 1975 1415 0794 8005 2344 7324 7054 7414 6974 5464 6144 4704 4864 6514 5324 3614 4884 3424 5114 2804 1734 0354 0184 0123 8903 8493 9053 8753 8303 9783 7563 7793 7403 5953 7583 6123 4913 4273 5713 5393 6383 4413 4443 4643 3093 3033 2543 2973 2013 2003 1043 0853 1483 1543 1593 0542 9572 9092 9952 9722 8772 9042 8652 7782 7782 8052 7752 8752 7592 7352 7312 6272 5932 5482 7682 6182 6202 5892 5352 4472 4412 5402 4272 4702 3742 3212 3722 4152 4382 2702 3202 3992 2422 2712 1442 1572 2302 1182 1112 0802 0652 0502 1042 1522 0442 0882 0082 0192 0802 0592 0061 9582 0321 9991 8452 0691 9931 8881 7611 7801 8161 8001 7671 7321 8021 7201 7441 8051 7661 6951 6741 7061 7421 6951 7461 7691 8261 7351 7091 7311 7601 7521 7821 6321 6471 6731 6691 6231 6291 6391 5901 6741 6281 5721 5851 5671 6001 5681 5721 5511 5651 5671 5061 4791 4591 4461 3731 4471 4001 3141 3801 3811 3741 4281 4251 4751 3941 4411 3511 4761 3431 4031 3501 3131 3991 2981 3351 3111 3791 3271 1921 2321 2891 2391 2471 2321 1681 1851 1491 2161 2571 1591 0681 1021 0931 1511 1041 1721 0641 0521 1531 0181 1061 0671 0261 1561 1641 1821 2151 1871 0351 0619851 0371 0121 0481 1011 0861 0419851 0301 0601 0671 0491 0239941 0221 0669631 0741 0401 0291 0561 0289591 0211 0429969499599609869979229609669719379809679649689219589469369369259729299849328919749268759339251 0059619489461 035931964863939874996912934842927897936867808864804746808764816817791755766847822803783794798773775788778777833784790771729796800818779760713767720728762756714691718737678688739641650717709674657749692695724640650682651619653630631646667611629652618617652617650640650638667650659627684613579634577595583552603614511557604576573569583542585571587568593558569582546586586574565643602623578566576547551506542585542573534512555531533559498537575511560522548521546548497525568509505515516525522520539505560491540532553518538490520532491536481528500444473513509518505530445442485511506450469483470454482495543544496568521467524479477498462469469474431433392413435428476428487458429426429420436454419472505480430429397455404446425437419427393404424422388406408391397390417405383373380391353416381408380389341394408409391364359392401344357406382384416442360406344353391381359374370364378335379386344319363338353310296286296326341316300307322325325302307342305358332321349320328329292304288341347324288290307279306288287285276283304306294288287301312272279318296315289336317314306302310306311314342319310308338325305347316333329299309311344280332327288326321296320323306269295284295306292291331328324322318302310319310312287320298306310353333326290289307311305318327277311288287293314326280295271278287279283301281314307300268281283288310296257283272248272291314306283279286292312252286271273283312311254271276266254282277270264262258273276299287283271274239247269282254263238280272265291304284256281284294259271282247252251255230235234227235252231244208228245233231247259271209241208199269245251252256287248239225261219239283261253236231209222223196232216213246302 781100200300400500600700800900>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.

4 225 6560000000172 428 2250001 652 058 586000000000903 288 0920000912 798 85000001 806 139 09900003 520 878 70200014 777 397 86000510152025303540Phred 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).

99.2 %155 954 94599.2 %0.8 %

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.

98.9 %155 598 27098.9 %1.1 %

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 %356 6750.2 %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 %78 639 78550 %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.

97.5 %153 330 55297.5 %2.5 %

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.

12.2 %19 214 73812.2 %87.8 %

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.

8 065 841143 24184 664169 719122 532129 479150 371200 83588 179154 61470 01863 07790 901102 53852 107121 55486 38897 932126 388170 545169 215172 676204 807171 913282 674464 83527 092809 88039 57938 99077 32182 11337 097102 54140 53241 36868 44689 00923 881134 9302 070 58298 13899 615156 454137 857250 138212 106353 881470 51874 44084 46081 39691 98455 43197 88994 46173 711225 24082 470143 234140 254 676051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M140M# 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.

99.77%99.75%99.76%99.77%99.76%99.76%99.77%99.77%99.77%99.77%99.76%99.77%99.76%99.77%99.76%99.79%99.77%99.76%99.78%99.77%99.77%99.76%99.84%99.79%0.23%0.25%0.24%0.23%0.24%0.24%0.23%0.23%0.23%0.23%0.24%0.23%0.24%0.23%0.24%0.21%0.23%0.24%0.22%0.23%0.23%0.24%0.16%0.21%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped