European Genome-Phenome Archive

File Quality

File InformationEGAF00000852740

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.

573 848 497283 323 148123 090 73661 910 67730 218 56817 201 81010 121 9386 730 3684 760 4713 662 8652 978 2992 536 1402 223 1271 998 2391 811 7241 670 5731 560 7431 469 8351 388 5031 318 6401 263 0681 204 5881 157 8231 116 3251 080 9621 048 0941 017 050989 950961 520935 983916 486895 328878 451858 870841 482826 518813 523798 355787 840776 607765 576752 315739 083730 112720 566709 766699 065689 766679 916672 965663 440656 778649 850642 309633 316626 700616 462611 543603 648597 671592 104583 534578 667572 314567 175560 377555 185548 210541 847537 457530 793526 522520 745513 557508 667503 607498 985492 714488 224483 554479 407473 162468 813464 588459 043453 826450 091445 216443 367439 687435 193430 595426 847422 345417 345413 046407 790404 853400 621397 192394 570392 405387 802383 323380 866377 220373 723371 016367 098363 228357 919356 333351 352350 297346 354342 540338 594335 712332 970328 742325 856323 121319 999316 379312 479308 523305 376302 025299 439295 814293 662291 887287 955284 786280 878277 212275 559272 436268 942265 923262 303259 363257 184253 145251 535247 998245 605242 363238 731236 311232 439229 962227 978225 168221 339218 807215 619212 276210 332208 485204 763201 662199 363196 338194 474191 178188 390185 826184 043180 208177 545174 464173 229169 841167 311165 438162 774159 995157 896155 093152 398150 607148 023145 715144 241141 216138 986136 791134 352132 489130 053128 149126 649123 972122 512120 470118 169116 202114 347112 420110 477108 763107 172104 769103 034101 05299 23797 67895 90894 95492 51890 86489 07787 81686 49984 86382 87482 29179 70179 52877 23576 36175 12673 08071 96871 22670 10068 68967 25465 79965 16363 41662 23560 78159 76158 73258 02156 78055 09754 35053 20452 08351 36450 13149 18848 31447 19945 91845 46644 24443 45742 77341 75541 02639 87139 19938 72438 14336 97736 34735 84835 35234 31033 81933 25132 36931 45931 13530 51529 81029 23628 54227 59627 66726 73625 92225 10924 84724 12323 54523 58122 72622 32621 94421 29021 04020 50819 59919 25218 87918 19417 85417 46317 46316 79516 36216 03815 63915 14115 00614 83414 33314 14813 65313 25513 01412 77112 25511 96411 68311 39011 23210 97810 49910 40410 2219 9539 7929 6169 3899 3129 0418 8828 6668 4198 3008 2467 9217 7387 5927 2707 2197 0426 8596 5596 4746 3856 3516 0635 8795 7945 5665 4205 3685 1085 1645 1284 8874 8764 7554 6764 5814 4044 2264 3034 0694 0634 0503 9943 8223 7683 7493 6383 4763 3903 3303 2563 2743 2723 0762 9672 8572 7932 7362 6062 6092 5012 4172 4542 3552 3752 2602 2702 1132 0612 0331 9682 0852 0511 9371 9211 8691 8641 8231 7261 7371 6171 6371 5771 5151 5241 4681 4211 3971 4381 3071 3101 2601 3601 2591 2991 3051 1511 1251 1561 0821 0351 0231 0381 0279759999709489669109598768939218198538048458048177997827387276786486967116456426456876296036295975845725545635095515274835165295175295114874985424875054694854754655204594123994124583954123693803423663473643603883203413663323553273183143213373053192963132983012973172893032632772942683132943032842932722652652752632672462522252382542492342512362332132252402272462082281892211982122292152081651821792051742182162102071931851831791932192121812022121871841811931731771951841581901841931841511672051721581791681761781681701701641751741781651371491531441701631501571501391651631641641411631651671771781321511681731291531241381031241501301401241441181291111131221061121351251161051131061369511110193125931049710110666909562907777799488738857767895917571727477726264846968626964656269587178844447525565736258595858583757575848565940584357545453695642515151515642464835403050294646332436343536404430354049343141443142424244473140313534384337403741383833472740402934313429264224323820162228273120322531243028222119232325253025181320211919182020342612262111161317212316231813191820231519151815231920231522172121142529182816222318162016181822201225181323131615191215181713111915178161491188811141417917161713171991015101518121114121789171320101415221612141816211612261919282117251417122414131918142115121319111017111512971018188171415189131517151118181113161511151012171516131611810129515133 819100200300400500600700800900>1000Coverage value101001k10k100k1M10M100M# 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.

2 728 268214 7563 527 6832 606 1404 568 0435 642 14010 850 46510 101 44715 655 83220 289 42712 750 51413 650 68111 010 95412 546 32813 974 36315 136 17217 221 07520 864 04919 153 45621 108 74022 422 85727 427 35828 527 95731 796 46435 851 16642 817 10757 117 88965 144 43299 528 612137 842 608218 625 140347 645 587510 151 176691 299 492999 320 6541 190 186 230860 103 519641 514 587697 451 187728 657 748697 312 877689 888 145590 370 634377 593 590105 226 051573 2500051015202530354045Phred quality score0G0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G# 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 %134 656 79099.7 %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.

99.5 %134 426 91699.5 %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 %229 8740.2 %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 %67 519 97950 %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.

99.3 %134 131 47499.3 %0.7 %

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.

17.4 %23 433 75117.4 %82.6 %

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.

3 505 7429 7305 96920 3356 95310 45811 23114 81413 71681 77633 65431 83773 89423 00118 620284 45323 558103 08653 89722 501131 1286 635175 282514 5876 11036 3055 3895 8874 4953 215 6406 8794 5545 0767 0825 2478 285352 8701 273 94615 40535 14830 36519 9338 27860 91414 47319 537263 70032 31533 81138 04265 77227 39274 96756 28679 236130 322192314 462295123123 498 94094222191268355781103 5170510152025303540455055606570Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped