European Genome-Phenome Archive

File Quality

File InformationEGAF00002144935

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.

1 395 4651 015 833896 823891 114926 9591 018 1411 140 9261 335 5991 584 2081 972 9092 536 4863 321 7854 439 0095 961 7218 015 55710 641 69413 994 45318 070 51422 959 62228 616 06235 007 74042 070 21349 588 40957 499 48365 551 93673 525 33781 197 04288 368 80494 802 884100 411 598104 928 433108 321 001110 542 173111 491 571111 319 181109 964 758107 593 635104 290 468100 248 99395 492 43090 283 36484 714 01078 939 75973 067 58067 172 83361 400 18755 782 22550 433 49745 326 23140 587 36236 165 01432 056 50228 328 38924 930 63321 863 50919 130 74716 676 84114 514 78812 616 83110 936 9579 465 3538 178 1397 075 0886 112 1615 286 0154 570 0953 961 0013 440 7002 997 5762 616 4472 299 1182 030 1401 797 7771 597 3191 434 4031 286 5941 163 8521 060 168971 353888 661822 168760 161709 197661 867622 070583 055548 493516 185488 767461 284434 925413 629390 979370 729355 717339 021320 407304 178288 105276 831262 383250 016237 589226 841215 941208 728197 717188 727179 674170 998162 944157 697150 849144 351138 767133 089128 598124 121118 855115 007110 853106 663103 11799 53896 91293 47289 85186 96385 01781 53879 00277 14575 06772 68471 19668 92566 68864 83062 93360 72159 08957 67355 78354 24652 33051 73849 47447 88946 46745 75444 65742 70742 13941 25140 26039 21638 83037 61237 61436 39235 59735 08134 81034 09033 32932 72031 83531 95831 39330 55830 30430 18930 28029 36528 77028 60827 89428 30128 20427 48427 50626 77226 33526 45625 67425 65525 31524 72324 60324 18923 86523 09922 82822 42022 20422 15421 69821 27521 43321 11020 60420 48320 29320 03619 27919 44618 78518 34718 28517 61917 46217 24116 98317 11516 54516 46716 40816 32216 43915 94015 72915 69715 53814 86214 61914 72514 32414 31214 17313 87113 50013 72413 68713 15613 14113 16812 77812 75112 68112 33412 42412 17112 17611 96712 03812 16812 05011 84311 74711 19311 26211 12111 17411 26911 07411 32411 01810 66210 41310 69910 44210 57510 14410 12310 0139 7239 8989 8199 6529 5959 4809 2779 2209 4039 2969 0279 0528 9119 0188 5908 4198 5738 5238 3048 4938 2168 3618 0768 0768 1168 0818 0107 7627 7787 5727 5997 3937 3747 2127 1517 2087 0327 0267 1047 0206 9966 9657 0707 1697 1437 0677 0007 1887 0626 9446 7986 9036 7496 9296 8696 8016 5906 4796 5106 5836 5716 3846 4096 2136 1325 9736 0496 0325 9615 7865 9165 6925 7865 5375 5455 6965 4235 5535 4615 4555 3965 5675 4055 2545 3575 2965 1784 9925 0765 0124 9494 8694 9194 8894 9104 9334 9774 9635 0134 9594 7624 7054 7914 6344 5674 3834 4154 3464 4784 3374 3354 1624 2354 1784 1594 0624 2144 0083 9423 9943 8983 8403 9133 8173 9213 8813 7233 7083 9273 8643 7003 8163 7073 5543 5763 6483 4433 5033 5263 5313 3843 4233 2783 3243 3053 4463 3283 1963 3473 3183 1373 2303 0733 2263 0423 1323 2323 1053 0673 1833 1443 0683 1243 0192 9072 8972 9582 9452 8932 9352 9702 8212 8482 8162 8802 9462 8502 7182 8462 7312 7592 7812 8282 7382 7182 7362 6532 5992 6582 5772 4662 5112 5352 5112 4342 5122 4292 4092 3622 3492 4422 2982 2892 3842 3872 2752 4042 2852 4872 3232 3072 1252 2622 1902 1512 1142 0492 1462 0651 9952 1182 0661 9322 0791 9961 9792 0771 9011 9991 8721 9171 7791 8701 8371 9221 8191 8551 8441 7531 8321 7541 8231 8171 7111 9141 8211 8751 8551 7701 8261 7921 7111 7031 6471 7251 8641 7821 7351 6941 7571 7721 7061 6111 7001 6431 8261 8821 6891 5971 6911 6181 7171 6321 6181 5711 7181 6591 6661 6431 5191 5821 5691 5201 5131 5541 4611 4291 4621 4391 4771 4241 4441 4641 4741 5261 4551 4591 4451 4141 4361 4411 4181 4641 4461 4541 3911 2891 3731 3971 3001 3291 3481 3541 2761 3291 2891 3751 3021 2951 3691 3621 2691 3431 2311 2481 2761 2211 2221 2311 2041 1731 2601 2721 2601 2411 2061 2751 2061 2581 2201 1341 1991 2221 2391 1981 2411 1741 1541 0781 1391 1571 2091 2191 1451 1151 1651 1911 2031 1581 2151 1681 1411 1091 1361 0671 0701 0391 1561 0711 0481 0309911 0721 0331 1129661 0091 0421 0271 0541 0311 0301 0629691 0319919941 0521 0059871 027990974939976976976952994986983939945963880934892909958894811923924921885907913881928935838791824879862834875801866852894869854840841895883881892883881857823806857792785773785764764822775767788764782731829883791714761738727718726750799740698817745785783759719738762716683678680707696676691668639628622647667620605629712689680702673721646663673691779629651631645638620622631658627674646669667672687681704697647612691612658660662647589642620638610604530616611588607614630649618610589573544614600635583579647607648564595557573568574552555539548533577562533568575580489552516504554538516515540577565542572521533507537544462491547505463501487519597559533551453469530485453479431467485475518482513487463498455448463441421422453421433448536458475444437467466449445441479485448489540481436452401383439408429411424434459445494392430413435422380415424438401426479414481439424391453380401435418406354397378384342387363386384374399391452384370371374381379392419404414418376371389410401421385396377413398406408402397392378381384352380333384372358549 418100200300400500600700800900>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.

0012 009 88600000170 967 8510003 523 444 9530000000002 185 740 66400002 660 102 00200006 087 583 437000013 042 224 95100083 811 519 85600510152025303540Phred quality score0G10G20G30G40G50G60G70G80G# 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).

99.7 %741 365 22799.7 %0.3 %

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.

99.6 %740 464 31299.6 %0.4 %

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 %900 9150.1 %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 %371 645 31250 %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.

97.7 %726 346 34697.7 %2.3 %

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.

16.2 %120 172 87016.2 %83.8 %

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.

28 008 5641 050 082853 3171 195 644716 781589 109675 128660 154304 372908 421562 966598 796713 856704 429383 392866 635504 707474 445599 583653 749470 136816 463742 867705 0891 066 2451 637 168146 2302 978 092206 924195 495322 848365 745116 769545 205175 196171 623304 860409 65387 344645 51010 287 557575 6893 294 975763 962532 789412 525148 640151 591523 611703 1003 915 9241 142 442866 117803 119725 6622 531 2861 069 496906 881769 196677 077664 034 614051015202530354045505560Phred quality score50M100M150M200M250M300M350M400M450M500M550M600M650M# 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.

99.88%99.87%99.89%99.88%99.89%99.88%99.88%99.89%99.89%99.88%99.88%99.88%99.89%99.88%99.89%99.86%99.86%99.89%99.84%99.88%99.85%99.87%99.52%99.88%0.12%0.13%0.11%0.12%0.11%0.12%0.12%0.11%0.11%0.12%0.12%0.12%0.11%0.12%0.11%0.14%0.14%0.11%0.16%0.12%0.15%0.13%0.48%0.12%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped