European Genome-Phenome Archive

File Quality

File InformationEGAF00003609391

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.

359 725 413260 343 719151 033 24598 725 40754 876 44637 874 82322 231 39016 554 84010 740 8218 531 5446 266 4185 274 2564 307 7083 802 7343 344 3753 049 1272 809 3502 608 2262 458 2472 325 0322 210 0392 122 0122 038 8871 966 9941 910 7331 840 2061 781 2121 732 5581 685 5051 645 2731 600 0471 563 7471 520 3361 496 7891 466 4231 435 7861 401 2341 375 7941 355 4651 326 3191 301 1711 278 4161 255 5381 239 0381 213 9921 197 6711 174 6791 157 7271 136 2161 119 0261 102 1761 082 9051 073 7731 054 4801 040 2231 022 9231 006 108993 342981 010964 927955 419937 620923 145914 484899 471883 984873 153859 635846 263839 894827 867816 879806 968794 592784 366777 621766 527756 162745 878738 623731 398719 428713 326704 109692 536683 682676 455667 269658 424652 444645 714635 867629 219618 133612 821607 551599 567592 776586 971580 198572 519564 885558 333552 673548 169541 673537 249526 271522 701516 746512 251506 592500 422494 367491 448487 319482 237474 409468 291465 279457 838455 096450 787446 088441 228436 486431 106425 610419 519417 326414 016407 388405 587401 211395 773392 523385 280385 355379 704374 877370 477367 301363 776358 647355 162352 311347 281344 498339 489337 950333 368329 795324 890321 704318 211313 867310 969308 064303 829300 860297 499293 309290 194286 322282 921281 869277 942274 348270 492269 416264 724262 433257 844256 476253 012249 813247 649246 013242 108240 016237 074234 528232 726229 069226 902223 276221 530218 253216 377213 993211 806209 617205 902204 816201 724198 793197 396194 878191 980190 772189 044186 733183 754182 135179 376178 282177 779173 941172 252169 989168 316166 201163 999163 246160 600158 925156 448154 808152 948150 863149 744147 298145 793144 665142 783141 161139 564137 828136 721134 950132 061130 860129 372128 598126 491124 205123 756123 522120 908119 780117 448116 638114 844113 626112 699110 797110 373108 627106 058105 553103 523102 898102 95799 89999 26098 74296 99696 23495 27193 18392 35791 00789 51188 83287 02187 09984 77384 08583 79782 85081 21980 88779 36577 73376 90275 92174 88675 01873 86972 90972 03871 14570 16269 73068 08267 83766 86865 82564 79263 90063 00962 42361 91261 21360 25760 29458 71758 47658 16557 43556 00855 12455 09854 03253 78352 66152 15551 60850 81150 09749 12948 35247 99947 40746 68546 16445 58244 76444 27543 37543 28842 93342 28141 42041 06239 94640 09938 97438 31437 94137 63336 98836 38735 79635 53434 99634 52234 19833 79833 51332 41732 09931 69831 14430 39630 25329 66629 16728 92828 35127 84127 66027 37126 72526 41726 06525 77225 31525 02824 88524 37124 06923 70623 87723 49222 43922 06521 85621 35121 06820 71120 41820 13019 73419 93719 43419 11218 81618 58718 33317 84217 92417 38117 30917 06016 84316 53316 33615 82915 86415 33815 17715 22315 13114 47014 47613 98013 92813 79813 57513 22013 16113 09812 62512 42312 24512 06312 02511 92911 88911 51611 34611 32610 70410 93410 60410 4979 96010 0609 9119 7239 3939 4929 3279 0368 8998 7308 4078 4388 3318 3688 0927 8307 9237 6817 7387 5817 5617 2797 0076 9646 7186 8886 6626 5886 2596 2166 2765 9745 9075 8615 8005 5355 5625 5195 6425 1815 1155 2625 1354 8774 8254 9674 7134 7624 5404 5244 4444 4864 3854 3384 3094 2894 0864 0923 9983 9883 8993 8943 7233 5983 4613 5713 4993 5393 3733 4413 1653 2493 2943 0783 0553 1412 9582 8762 9132 8392 8672 7602 6852 7102 5772 6812 5682 4922 5682 5122 4152 3352 2072 1662 2142 2622 1262 2452 1002 1672 1162 0731 9811 9401 9011 8651 8211 8711 8881 7981 8001 7141 7291 7021 5971 6241 5671 5301 5491 5091 5211 4521 4181 4031 5101 4491 3831 3601 3151 3221 3391 2681 2441 2061 2291 2201 2321 1661 1891 1291 1491 1071 1771 0881 0961 0671 0761 0551 0271 0121 03295699289593491292184486599784085481676481176778981577373273174673468770071667970067967266964468664768165869162760862161061659459357959456152357652655654855353864151048049748050649046044543947149147146846746647145345842442845745245341847041041942639840540540142240839038739635740939747338641339136239933936036336435132635336731433534131134031735034032434931836630734531233032034027632330728428930630026327226727423929926423326125226123826125224420821124123823922324223823623320822721820519722421220720321519722422024320621018223023321919721422521320918720520019920921821520321520422719718718218618920723322620019620122420419122319518921721117619221218017018118515918421318618118519216616618518119319520219117621718818216516617117817120817418418219714714415514217517016616117419217717817817518017020319218418028221520618718817017719718519216115715215614612814015013016212114913913914114712926913413312614512514311313214613514114513913313714615813313212713314513212614012415111411412413211311416415711412511510811710611910710711711696881151131051009511491108109112861201041161171171171151051321001111121041051062139110210185989210695986811293747985103991019086681017782798274202908178725371737471608673766065787479617271595964806958676755556719 814100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

448 4290000000000539 888 7020000000000000831 591 0000000000000015 461 717 66700000510152025303540Phred 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 %111 084 58699.6 %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 %110 909 69099.5 %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 %174 8960.2 %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 %55 740 54950 %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.

98.7 %110 012 35698.7 %1.3 %

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.

49.2 %54 856 15349.2 %50.8 %

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.

2 209 88520 59412 62935 31115 77717 01922 87242 86523 37635 18910 0478 71214 66714 0386 78529 2588 94310 69715 85717 70616 40733 65926 91923 01740 27998 4674 419498 8905 4835 22714 58012 4067 05927 9645 3185 46711 39315 9043 55740 830572 90323 06015 87236 98821 24664 05364 009140 142373 15115 49930 00823 63237 97211 14620 87521 86416 376127 08017 27944 144106 613 728051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M# 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.

99.84%99.84%99.85%99.86%99.85%99.85%99.85%99.85%99.84%99.85%99.84%99.85%99.86%99.85%99.85%99.83%99.83%99.85%99.81%99.83%99.84%99.84%99.13%99.74%0.16%0.16%0.15%0.14%0.15%0.15%0.15%0.15%0.16%0.15%0.16%0.15%0.14%0.15%0.15%0.17%0.17%0.15%0.19%0.17%0.16%0.16%0.87%0.26%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped