European Genome-Phenome Archive

File Quality

File InformationEGAF00004839426

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.

66 812 294125 789 043192 360 617254 851 046300 989 666322 392 614318 036 100291 819 216251 512 986205 112 493159 263 404118 533 38384 881 38158 760 74839 606 17626 030 89916 811 01510 753 5966 844 5804 360 8592 832 3711 883 5001 300 682942 713712 063570 584467 150397 626347 939307 570277 745254 681228 146207 247190 968177 284164 609152 148142 475134 620124 771117 442110 172103 85298 23490 81185 62080 40874 80870 99767 73662 34760 22157 37354 47051 29749 12646 68243 96542 18740 42839 02338 05835 64634 70832 66332 17930 57629 43628 17427 23026 47125 85325 04124 26223 17322 29822 29221 73821 14120 26720 03418 97418 54517 95017 71817 29816 84516 56016 17315 79714 83714 35913 75913 67713 71013 72813 48113 16912 88612 60012 43311 81611 79811 21010 84210 64110 52310 45910 26910 4379 8659 7539 6709 4509 3229 4079 3469 0398 8198 7978 7968 6498 3928 1258 2238 1037 8917 7217 6167 9457 6207 3777 5587 2317 0477 0896 9756 9906 9946 5726 7836 6396 4166 2956 4586 4016 4486 1726 1286 0506 1766 0815 7195 8435 8085 8345 7705 5595 4555 4165 2805 2735 2785 2785 1045 2815 1305 1254 8884 8434 6844 7384 7144 9434 7484 4824 5974 5344 5734 5374 3104 4734 4984 3644 4264 1254 1104 2394 2383 8983 9113 8613 8983 7823 7273 6463 8023 7393 5613 5783 4843 5633 5303 5243 4823 3723 3513 3273 3403 1603 2153 1483 2483 2233 0173 0553 0263 1223 1212 9563 0943 1063 0723 0332 9332 8452 7172 9502 9112 8422 7202 7182 7562 6552 6222 4222 4712 4792 4682 4942 3472 3602 3592 3772 2722 4392 3692 4052 5082 4902 3292 3232 2752 2432 2222 2502 2452 1802 1382 1502 1322 1201 9932 0881 9822 0552 0772 0772 1022 0792 1561 9641 9622 0122 0081 9181 9281 7941 9011 9551 8851 8001 8201 8381 8961 8731 7671 8971 7171 7291 7531 8301 7591 8941 8331 8501 8391 7811 7651 7771 7841 6701 6101 6051 6951 6281 6511 5691 6811 6951 6511 6311 6181 5371 5081 5081 5681 5851 6151 5751 5941 6271 5141 5301 5731 4951 4881 4671 4951 4601 4051 4241 4651 5061 4731 5201 4761 4931 4641 4771 4611 3551 3461 3881 3381 2661 3401 3101 2651 2671 2961 3401 2941 2971 2941 3021 1951 2841 2021 2811 2671 2211 3571 3101 2831 2901 2191 3221 2621 2291 1811 2451 1861 1891 1521 1331 1561 2271 1781 1711 1821 1051 1611 1241 1711 0851 1791 0891 0411 1081 1261 0821 1521 1421 0951 0721 0781 0831 0521 0721 0511 0091 0051 0201 0521 0441 0579841 0119931 0209679701 0049919999791 0271 0439329729851 0329891 027895985910909888876884923920928927886961974905875840829871840851819802805801833828786839847869858858930838844943934901939891865873850813859820849873856851888877853880817867886848864890821810806814851822833830896780741790729728786830767772735800734759785758758762744781759745744728733730713709751714706732702760756747699711697700769692675797764719689704689723738664675609680623638589604660606656616594636625602599645625639618627616585592611545548562572583576690619616620661591655657656616586598559601568603609654632608611536524552561563554504522532479489484448487469478546520532500552527521513497431531476495508474467473439456510471430426482474471492484514441509501474454451462439458485453474452479445463416437463441472435436449447447437423387395423429417421433428413444429422434428428477447442450403427454432423393397418421410394414399410417447456446453407456428441446419432448445457491483442473448484472455469484460391444463450488467426498470423442465453417450417419454406432427444443434456443470472450420444426415463425460426414432426491409408392411428427360362399389405384403402375427408408374424372401408372406375383359396419376405387368384384432383354391364380394409364403362421409370364385393426410434379410396398398401526438375411443420384426410420411456413436370382377400377357395408403397396383410442428427362400389368364329359363365381346379384370323349354368291322316345326324338325317347317339350341324332347342347314320321334334335318328347332316340290311335293333326327332310303313342317307339312327320291292309284294283310314322298283285286321285327298305306297315308263267307298257301313338298293295284245270275287260277261268270236258230202219267253242248257238233245228234239230228267235248226214238240225279 088100200300400500600700800900>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.

4 165 467000000035 478 236000999 011 655000000000574 367 9520000645 426 02400001 414 480 93500003 009 663 31700015 716 593 90400510152025303540Phred 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).

99.6 %147 786 22899.6 %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.

99.5 %147 563 40699.5 %0.5 %

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 %222 8220.2 %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 %74 169 49550 %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.

97.4 %144 521 03897.4 %2.6 %

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.

10.8 %16 002 17910.8 %89.2 %

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.

7 183 338138 17385 833165 818122 094128 930145 381198 12197 218155 90773 29863 55185 00698 43452 223118 39684 31794 028117 437164 408166 616168 007207 870163 841258 696417 64130 670720 97143 38940 01873 15481 43040 051102 62340 88042 01663 52685 04025 419132 6831 732 95792 40190 596151 775129 505237 929203 366320 008485 48462 08376 08468 94384 55743 51092 79279 26258 486197 50463 215121 110134 126 285051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.85%99.84%99.84%99.85%99.85%99.85%99.85%99.85%99.84%99.84%99.85%99.85%99.85%99.85%99.84%99.86%99.85%99.84%99.86%99.85%99.85%99.85%99.9%99.83%0.15%0.16%0.16%0.15%0.15%0.15%0.15%0.15%0.16%0.16%0.15%0.15%0.15%0.15%0.16%0.14%0.15%0.16%0.14%0.15%0.15%0.15%0.1%0.17%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped