European Genome-Phenome Archive

File Quality

File InformationEGAF00000644580

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.

315 103 04158 093 09713 520 4266 811 7284 712 7483 859 9333 359 7402 989 3452 705 0482 485 7952 300 6692 159 4542 035 5791 928 3341 833 0481 748 2651 674 8201 608 4571 551 2481 494 9411 449 7151 403 5831 357 9891 316 4201 278 4761 240 1151 206 8401 174 3611 146 3071 116 6331 089 5391 058 3311 032 3141 009 874987 452965 585942 611921 232898 130878 474855 245836 811819 020800 592782 461763 948743 908727 035708 657691 507674 028657 876643 733626 980609 936595 819579 349566 715553 209537 986524 143512 200496 718482 070469 821459 330445 079434 886423 732411 416400 861389 523379 572370 077360 629350 285341 320332 707322 984313 664304 669297 195289 871282 557273 299266 669258 770253 206246 607240 298234 254226 999220 977214 650209 320203 835197 685192 320186 189182 782177 339172 610168 751164 786159 563156 168152 616149 107145 519141 356138 903135 346132 196128 958126 505122 813119 797116 579114 055111 384107 715104 639102 209100 26697 25795 02392 70690 53088 10486 47484 40083 17480 58479 46177 21575 15672 92871 66169 39068 15766 72665 03063 74562 65860 82559 42458 25357 19855 25454 51352 94251 48950 73849 89648 77347 38547 09645 80544 69643 78743 01142 10741 01040 27839 30638 61038 04437 34636 97936 06035 32834 52333 39833 02132 39931 72031 02130 60530 10629 52028 91028 50027 52927 28426 63426 03125 65524 69424 59124 08123 61223 12922 49221 95621 59721 09920 59320 08419 66019 33118 93418 67518 18418 21417 74517 11517 14016 58316 17516 13915 78015 57615 11714 84114 61814 27114 01613 86413 65613 32513 24613 09912 82812 57712 15312 07411 71211 68111 58411 37811 12211 17310 79310 67310 55910 26910 1549 8659 8899 4509 2499 0189 0128 6458 6878 3718 3448 3938 0977 9217 8307 6557 4757 5457 4567 2977 0437 1216 8796 7786 5876 5206 4706 3156 2796 0416 0245 9375 7595 6505 6875 6785 3665 3455 3615 3145 2085 0895 1104 9664 9264 8304 8814 7494 7004 6284 5584 3524 5144 3534 4824 3174 2254 0354 1544 0163 9363 9473 7583 8293 7753 7933 8343 4303 5163 5073 4483 2533 3643 3593 3143 1223 1523 1062 9882 9902 9052 9502 9342 9742 7692 7192 8102 7232 7392 6282 5842 6222 5722 6222 5612 5622 5632 4172 5822 3212 3712 2862 3392 2312 2522 2452 2002 0772 0842 0852 0121 9141 9181 8671 9481 8601 8561 8841 8701 7361 7851 8131 7461 7411 7081 7271 6591 7351 5751 6211 6011 5881 6161 5891 5161 5401 4931 4901 4621 4061 5001 4371 3511 4911 4281 3531 3751 4361 3301 2521 2621 2691 3201 2711 1991 2091 2471 2331 1841 1941 1811 1821 2271 1461 1511 1501 1781 1321 1101 1231 1021 0691 0151 0721 0891 0201 0589969901 0069491 0181 0029349869148839259349278828927948318588029028328578588868618508428088707738147818677537847647597717427937647677977787837637807307037687447677016537526877066786966906766796156556916386616386125656276556086055986115816036375195935115835825255795455605295365445595085404944615385464845125104874724765424914814955044614854404414914304294584084014634374134084423684123853913993963853733823983603943783803903534143843793804263433904143793553663513783633803313193493683553213483263472993083153443223303123052962892883262803033162973132592922853013053152803123103043102973062883202892892863012662862762732793263032933192822822612323142903142823062772412522812582852512702952812552372342662312262402282352122182142332402232252342232112302542512201961961952002101921901971881701741621691631961751681581621711881871741421561651851791901771811721441851801511601501711681741421451781571761481601391451751481541701411451581391611411331461481421611291281351361291321331431431361511311141221121301221171221201321071161361311391161131121121381311181061121161161351171251191421381141119311412011912110910011394123116107107951151081311121061081061011181299712010710911111411492981001139895911049287110818893878176797986879788727494908196101948710582103761139710190838390726879867885848784667971818297938498947396868585917884961067293817289881007081988573807579828283828780767087696884706463688178917795898175748782681007971636081507964685954636274726161677886735665878278716482726159627661748580626868666558785662706265676255708556696848525048655247637457686353596055675736765917 865100200300400500600700800900>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.

2 340 229110 202260 0751 026 9061 808 5741 262 5773 910 8955 064 3271 604 5686 521 89421 181 13214 291 9775 641 4543 579 4612 141 6844 239 2931 911 7695 347 96012 345 26831 930 65119 679 79237 282 28821 043 47515 068 69612 700 76419 013 89211 012 77121 631 02118 299 63440 717 18585 096 46658 749 40478 731 14592 838 726135 968 355244 412 748302 729 671657 869 705979 794 0461 184 626 109332 251 181112 544 13815 830 53003 536 4620051015202530354045Phred 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).

99.2 %61 206 93899.2 %0.8 %

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.

98.9 %61 032 30698.9 %1.1 %

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%).

0.3 %174 6320.3 %99.7 %

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 %30 852 99450 %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.

98.8 %60 939 35298.8 %1.2 %

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.

3.6 %2 212 1643.6 %96.4 %

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.

6 122 7005 2024 8918 8114 42712 83412 35824 82219 86658 69649 35513 422108 20719 31118 086245 75441 041133 18185 51534 276175 7461 721114 719395 1041 51621 8502 1091 9172 1111 796 7135 4804 5405 7547 5747 77010 586248 516733 56920 3185 19436 41419 7022 42255 5322 4904 636156 7424 1229 2706 88618 6668 52630 62829 91848 29689 214217 65450 379 308051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M45M50M# 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