European Genome-Phenome Archive

File Quality

File InformationEGAF00000101359

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.

232 088 66848 108 35812 416 0747 078 5734 850 6244 009 4133 452 5343 080 5662 786 2122 568 0962 382 4962 231 3932 106 1201 995 2251 897 0451 804 6231 730 0641 662 6231 601 6301 549 3901 496 3451 447 7801 402 4011 365 8231 327 2321 288 6701 253 9051 217 3051 185 6151 153 5501 126 9771 095 3221 070 4421 038 2361 013 775986 057959 758936 548913 631887 095866 569844 832821 383797 874775 757753 464733 150708 436689 472668 058648 671629 593611 971591 667574 687557 636539 182524 808512 985494 032480 694464 573451 619435 341422 206409 010396 606382 776370 401359 939348 188337 157328 985318 919309 396298 713291 801283 041274 574266 665258 187251 724243 768237 367230 401223 817217 677211 523205 070199 052193 201188 453182 765177 724173 105168 527163 890159 684155 682151 320147 700144 764140 014136 361132 338129 448125 283122 689119 535117 033113 726110 576107 675105 323103 662101 22798 63295 61793 34791 32588 99886 80685 50284 02781 33879 81977 88576 62974 73172 94070 92269 74568 12266 50965 02063 56562 09460 54259 80058 40157 00356 10454 98253 51852 61051 20550 69949 19748 23347 90546 54845 66645 04044 12743 53042 60242 03740 60640 32539 39538 09337 86136 96036 30335 45134 77833 73533 21032 40331 92131 07430 75229 90229 60429 32028 56028 07327 27627 02526 64726 26325 83924 73624 36324 04823 71623 44122 76622 44521 98021 56121 29720 69220 16519 87619 65619 28018 97618 86218 70018 09617 68417 27817 03416 79216 49816 28715 93415 56915 50715 21114 96314 84014 31314 39914 01513 75513 45813 23312 93112 43212 62512 59812 27311 99611 94811 62211 30011 07110 85610 91310 38510 34710 1559 9639 6519 7399 5349 2869 2919 1118 8608 7928 6868 4008 3468 1578 1197 7917 7377 7517 4087 4367 2347 1746 9276 7896 8736 8706 5216 7046 2796 3806 1966 1136 0376 1605 9885 8345 8085 7245 5425 5335 4435 2945 1945 1545 1044 9874 9474 8304 8264 6164 6654 5604 5664 5064 2654 3854 3624 2734 1994 0474 0504 1423 8643 9303 7773 8603 7643 7583 7403 5543 5343 6193 5493 4353 3613 2723 2753 3023 1033 1573 1253 0252 9982 8382 9112 9842 8882 8642 6852 7552 6782 7732 6782 6392 5592 6162 4372 4542 4202 5132 4732 4552 3732 3702 2882 3322 3672 2652 2362 1892 2782 1592 1932 1292 1752 1152 1322 1082 0452 0172 0082 0021 9431 9121 9171 9191 9091 8351 8431 7781 7151 8111 8061 7291 8131 8491 7921 7211 7481 6551 5581 4921 5841 5911 5511 5531 4951 4241 4981 4141 5101 4831 3801 4041 4181 4301 3921 3981 3791 3371 2911 3721 2631 3121 2811 2001 1251 2071 2341 2321 2581 2371 2051 2641 2541 1871 2761 2251 2341 1421 2281 1701 1441 1411 1521 1681 1391 2031 1331 1541 1501 0981 1111 1111 0931 1021 1111 1301 0231 0221 0371 1031 0211 0231 0051 0401 0219819751 020970950954898905900903890914848856918914846909875861777812811806785815790798768711800756732712740735753670722719671656713717707664675659676628638619583592583607587575549575566561582557587598515546517533550507515516520501562467539484508517518513511491523444476427446427451395491444482451450430439402440442454436426403453443446413428411389362398383377389389426427378404409389369389392410414404391396420392395396361346380339329327365320357333369317318301324303321314317302299340299320328297324312315301318311314306295308314320298297307285285309288291293284244269295313286253251292268281265251262264274262286277284268276309265264257249273250225273235276265269250264260242229272264272225257265265276249246244224269271250245240265227224229273201217213234257252240246219230222227241201230242214199233219213217204217208185197194222211192220188209209212210193221212197205195225174175207200207196239192196193224190171188153189180182206180172185200173198170177178167172194165168167194190155190202162166140151152166157170137143140131132147133142151153133131164153144130139150172149134134135151138161164155146133148127154152133130136151134151150153137145148146140140128155136146136135134126131119127124115132119136108131134106137123132941261171291421191241321271271101231261161241031171291191031028697102117114126108121113951137412210111111810710312112410411096115129114112109941221078511412411210911712211511810910710311111010611310984105115120941141161021041101128911892899311611995849495868489929788837074867886808292749592101968791917810697818181648692836379808194709577687271100687360735968705867565165697516 593100200300400500600700800900>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.

1 133 282455 741595 0151 251 4153 250 7587 562 02710 630 34419 999 89538 202 89922 303 17410 788 3137 653 8478 798 22019 756 68160 571 65935 301 42118 995 89827 520 94418 639 18719 578 34521 400 12827 122 20042 751 82032 501 14337 787 01555 442 06251 927 94445 125 14559 442 29753 143 61460 830 47573 682 45493 490 014119 015 702143 846 176182 146 155214 351 223250 359 287351 797 212407 024 397375 311 145494 866 576383 130 028168 519 319133 749 51455 146 82061 125 48512 418 0514 753 61615 686 868005101520253035404550Phred quality score0M50M100M150M200M250M300M350M400M450M# 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).

97.2 %56 491 75497.2 %2.8 %

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.

96.6 %56 163 59896.6 %3.4 %

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%).

0.6 %328 1560.6 %99.4 %

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 %29 072 55350 %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.

96.5 %56 089 12896.5 %3.5 %

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.

6 %3 510 0586 %94 %

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.

7 585 9495 1634 8748 4884 58012 51412 35125 58320 55159 87152 03012 514109 58220 12717 972257 39441 459318 82381 84630 691164 0561 593105 219364 5461 45439 9772 3672 2752 2012 770 0306 1045 1086 2288 3928 19411 618229 547700 16920 6025 48836 49819 6562 37453 1742 2545 652144 5424 43613 8386 83238 4309 37633 15431 30448 64088 414207 96644 261 036051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M# 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.

99.24%99.51%99.65%99.31%99.53%99.67%99.29%99.42%99.32%99.33%99.67%99.76%99.68%99.66%99.17%99.05%99.5%99.63%99.66%99.7%99.55%99.37%97.64%99.51%0.76%0.49%0.35%0.69%0.47%0.33%0.71%0.58%0.68%0.67%0.33%0.24%0.32%0.34%0.83%0.95%0.5%0.37%0.34%0.3%0.45%0.63%2.36%0.49%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped