European Genome-Phenome Archive

File Quality

File InformationEGAF00000835450

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.

2 444 3281 795 0241 598 5771 517 3231 478 4861 478 6441 526 9681 621 2201 793 4832 100 0312 559 0713 237 8764 167 2045 386 9616 845 6068 506 73210 258 86011 981 00813 510 86014 758 79815 669 95816 291 41416 701 21617 047 11817 539 26018 404 45319 911 43222 299 35025 822 90330 570 48236 648 69944 107 78052 825 07962 570 54773 114 16384 078 32095 030 124105 510 748115 050 456123 181 824129 488 404133 695 475135 603 615135 161 850132 476 165127 688 991121 040 679112 864 423103 648 84093 700 68083 471 74573 246 61063 354 11154 028 83445 458 78637 753 65230 947 96425 080 52220 085 33015 920 88512 495 9039 722 3547 529 5385 801 9594 457 9343 428 5692 653 3082 071 9091 644 8221 329 5801 097 972927 165807 606716 734651 771601 371565 726536 769511 697491 853476 645462 499449 731437 039425 352414 383401 796391 962379 345368 988357 319349 116336 989328 258314 529304 125292 298282 153270 002261 590251 204241 664232 529223 931215 501207 083199 618193 254185 596180 026174 504169 465164 418159 534155 843152 408149 222145 709141 827139 449136 474133 568130 939127 825124 348122 820120 288119 959117 531114 612112 106110 650108 799105 430103 647101 20599 37397 41595 01593 28790 89489 37687 34185 44384 15683 03280 77779 79878 52075 87875 09573 49172 14671 51269 49968 94267 02665 09363 97462 69862 27160 33759 03757 83456 20155 69054 81053 09751 76151 39550 59550 28249 00548 06547 96746 45046 43645 35744 52644 00443 19342 70441 84441 02140 60539 92239 36039 35938 45138 61837 42036 92236 14635 82635 20935 00634 27133 78033 69032 63132 29631 89431 56030 99730 53929 85029 59029 20228 61528 38328 23827 93027 31327 10527 03326 22626 07426 19326 09625 74825 33724 94224 55724 22124 18023 85423 61123 33322 99422 79122 43722 21721 93822 01421 59721 58020 76420 73820 49220 08619 92019 98919 66019 65419 51418 87518 68918 52218 45518 26617 69717 93717 74917 38917 06516 99217 07716 78816 31816 20816 03015 67415 71815 81315 29015 42714 97515 21114 78014 88314 88714 46214 39514 29113 91613 63813 57713 63713 47713 32413 05712 67512 63812 32312 34412 15811 91811 79411 64911 57211 47211 27011 29111 22910 98610 59310 51910 49710 53310 22010 4009 91310 0439 8409 7489 6529 3889 2619 3059 2409 2959 1669 1518 9678 6978 7268 5598 4418 4098 0507 9257 8177 8847 7487 7547 7437 7257 6197 3127 4227 3377 1927 1527 0656 9647 0416 8536 6916 8076 8096 7556 6086 3486 3666 3416 3706 2416 1136 1876 0266 0446 0486 0365 8486 0255 9556 0426 1486 0455 9285 8715 7795 8625 6595 7665 7045 6635 5835 3215 5065 5115 4185 3365 3315 3415 1135 0685 1815 1065 0734 9904 9304 9154 7774 8404 6714 5714 8334 7064 6804 7044 6594 5274 5304 4474 4784 3324 3244 2594 2154 2964 1934 1814 2244 1894 2464 1504 0184 0094 1184 0643 9823 9273 9043 8873 8353 9173 9543 8973 7803 8713 7483 8003 8243 7493 8493 7203 6453 7423 7153 6343 6703 4673 6083 6303 6503 6863 6123 5163 5033 3853 3843 4533 2563 3253 2763 3693 3703 2453 3063 0563 1893 1783 2773 1693 1892 9963 1573 0283 0282 9803 0232 9472 9723 0353 0613 0353 0262 8972 9282 9152 9692 8632 7852 8352 8562 7392 7372 7072 7062 7502 6672 5902 7032 5212 6912 6482 5962 4502 5702 4742 4242 4132 4772 5112 4512 3492 3562 3882 2512 3192 2552 1882 2322 2872 2352 3122 2432 2232 1932 2092 1372 1412 1282 1322 0362 1202 0351 9882 0192 0351 9401 9501 9971 9831 9491 9341 8811 8581 8611 8791 9111 8051 8701 7991 8341 8161 6601 7161 7271 8111 7801 7151 7771 7391 6981 6951 7651 7001 6571 7841 7671 6511 5831 7001 6851 6961 6061 7401 5831 6101 6111 6721 5311 5341 5101 4931 5401 4411 5121 5151 5591 5471 4231 5381 4411 5741 5421 6291 4191 4471 4241 3891 4041 3961 4131 3741 3901 3851 3631 3691 4371 4791 3861 4911 4051 4221 3581 4321 3921 4561 4121 3961 4601 4351 3781 3491 4001 3691 3311 2621 3631 3281 4351 3011 3161 2521 2081 2311 2851 2481 2821 3441 3341 2041 2401 1571 2011 2581 1541 1941 1621 1671 1401 1391 1851 1641 1601 1511 1681 1641 1181 1961 2161 1971 1651 0991 0941 1611 1621 1591 1041 1111 0561 1111 1411 0781 1021 0851 0691 0311 0441 0279999961 0339779691 0089581 0059941 0011 0059799711 0379451 003923961955949943934898897917904892838869992992949908881885932919813817853828861814858830851890857850815843841818831836852855844777800843813766759793841783761756810828800747719807753738753766826787783679751765695706743679715745657696722710705711796729787732720729755802725706731736781714758716725766752746754716744670743781783705761724779692703712667689638624668678651670703620600592651701669626666700692660662644590639681636712638629603656599655621616590669584618637570625592578610599597549589580577622558595572611569631575539555571569578538560544552567583553549510532534535507525506568560531562536473525531485453474477513504469461475469491413461483472460461479459496474488519475477482496451490487450471533484518475504473484436509439490494450472493473454499447476447456462511482475484420455477453442416414418439452439455431415420381436432435416443406408437402402407425434434390377413430407457444416392415405380395454398425431423438427371400442428419450436377402396559 336100200300400500600700800900>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.

31 780 4560000209 831 21568 612 868739 723 531371 738 720116 529 302305 986 273115 103 15783 780 817160 465 77348 912 881181 400 200116 643 385180 495 969392 099 437255 794 458357 448 207147 751 778263 666 107378 591 295509 163 243675 821 490919 185 635852 304 713678 025 5351 434 576 1491 738 231 3803 195 303 7332 389 551 0583 518 139 8579 604 041 65413 561 266 4287 287 825 81112 633 717 0279 229 457 40714 493 730 81118 860 269 58725 945 149 45300510152025303540Phred quality score0G2G4G6G8G10G12G14G16G18G20G22G24G# 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).

95.7 %1 263 430 56295.7 %4.3 %

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.

94.5 %1 247 239 21294.5 %5.5 %

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%).

1.3 %16 191 3501.3 %98.7 %

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 %660 260 58450 %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.

92.7 %1 224 342 02292.7 %7.3 %

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.

2.5 %32 558 2062.5 %97.5 %

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.

114 481 6546 050 167429 78613 090 482376 358174 099234 19990 86370 12856 05313 028 70756 09941 40429 91431 56725 34425 06726 84923 49632 26127 20531 92644 30736 78948 55039 44448 37363 73077 39589 107121 403137 024159 833224 504292 223435 050455 148554 150860 0631 021 607736 820982 637846 969809 2631 023 2601 392 36686 26972 95762 00956 29447 28042 50737 11731 29828 96028 22027 80126 63126 79327 7771 160 985 612051015202530354045505560Phred quality score0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G# 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.

99.08%98.71%99.27%98.71%99.25%99.32%98.71%98.81%98.8%97.59%98.59%98.96%99.39%99.08%99.25%98.35%98.8%98.07%98.07%98.65%97.44%98.55%91.86%99.47%0.92%1.29%0.73%1.29%0.75%0.68%1.29%1.19%1.2%2.41%1.41%1.04%0.61%0.92%0.75%1.65%1.2%1.93%1.93%1.35%2.56%1.45%8.14%0.53%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped