European Genome-Phenome Archive

File Quality

File InformationEGAF00001404693

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.

503 181 076243 431 82596 771 84946 708 31020 733 47811 970 5456 893 9034 867 6153 614 5242 964 1402 526 7672 237 1962 022 7311 860 5381 730 0161 627 5971 538 9451 458 8621 398 2451 344 0921 290 8741 246 4431 204 3411 168 0991 133 9641 105 8471 077 2231 051 1271 028 3331 004 493984 566965 771949 953933 293914 843896 526881 749866 151853 494840 723825 025813 548800 413788 377777 783763 315751 798740 313730 029720 273708 698700 085687 071679 162669 718661 055651 564640 754634 889625 456616 567609 923601 327594 209587 825581 361573 181564 428560 369552 970550 087542 001534 199527 550521 743515 260511 576505 542497 631494 205487 295483 073476 548471 661465 363459 464454 375448 758444 611438 633433 678428 669423 425417 586411 470406 642401 153397 868391 262388 389383 083378 303373 868368 843362 504357 791353 717349 192343 171338 195332 863328 336322 089318 611314 114309 383304 277298 760294 545290 562285 853280 699276 218271 684266 681262 200258 510253 993248 940242 870238 746233 233229 688224 800221 218216 670212 420208 047205 191200 652197 167194 383190 235187 269182 947178 970176 089172 233169 201165 597162 891159 585155 905153 141149 216146 198142 475139 827135 947133 435129 034126 845124 951120 934119 045116 642114 017110 896108 741106 095104 280102 17699 14496 47593 84192 30189 06687 41284 86082 00880 33878 54576 43274 56172 85770 71169 04566 90265 26663 59061 94860 12259 33857 49255 38354 09352 34251 19149 46848 56847 11645 90544 35143 72442 16441 11239 92838 43137 54936 61435 51234 62833 54132 52531 80630 94929 70128 96128 22327 37926 33225 88724 94024 53923 58222 86221 89421 34520 69620 29719 26518 83518 25617 72717 22216 57416 19415 54315 00714 75714 28313 81513 18112 74312 37311 94111 61811 23510 99010 68910 2839 9529 5479 3899 0488 8578 6078 2738 1247 8567 5757 4597 1236 8546 6016 4946 4576 1365 9655 8295 4685 5045 2635 0824 8514 7494 5974 5534 3824 1634 2044 0323 7983 7543 5683 5423 4533 1873 1913 0562 9872 8342 9022 7532 5802 5782 5232 4532 3292 2962 2542 1582 1912 0342 0172 0471 9221 8631 7471 8061 7791 7201 6851 6141 6351 5651 5081 4521 4431 3891 3491 2901 3391 2131 1251 1471 1561 0991 0261 0121 0489859928829349079238889399508148877878438117857777187227156946596646637056086345605986315685725825455795935375694745134644784624594624694324384644304014224114033993753843623843813623993673503443173423203572803102903213412852862902792532572512482302532622322282202752222542402482442192312342582182442202182142201762202382052162342122262322172102351981861951971811781881981841791811741921841691681781891441661461501601561281661431391091151171031411501241051351321061141041169410010195112105819179757469939110777909677747780807860677875718860615162645964676259605165518078587372658758597650805657787175605567626461795871455158595765535259575154515131515051475161615956455351535349415153515448453347363936293739374534313146383641294442283237403533353531303644374429263417292532292735242927242725263229342226133031301929263223281917183827252624231622181918251916162214141313131116181517161617101215191414101720121991210201216141418171512171215117101112158148151413111416111926917151017131719232116141915161520158201916143214151291011121514131513121113151613181871612131741699212318179161319171513119121114871010107141361114151014891312101188137139710699681110811871021176768151210913416810659881141591099912177121213139161416131418171412151416131671315101113171311151091578577105691097865552547655126108778786887972356564697554343694532643226454272873106119676666458691 413100200300400500600700800900>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.

3 499 020393 1431 164 9111 033 9121 681 94312 791 9053 702 7513 797 6425 916 6296 052 6027 884 3746 164 0236 734 6057 697 6209 701 5187 442 7346 931 9879 181 0809 652 2309 432 66911 660 27514 774 80518 081 16719 957 91021 559 44825 385 29754 560 013104 404 034185 389 370289 646 935650 471 1571 380 017 350988 533 6081 019 674 2881 176 571 7241 095 809 314460 919 798167 279 00513 007 1540000510152025303540Phred quality score0G0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G1.2G1.3G# 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 %103 970 99099.7 %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 %103 798 94299.6 %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%).

0.2 %172 0480.2 %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 %52 123 73350 %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.

99.4 %103 584 86099.4 %0.6 %

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.

21.2 %22 134 15621.2 %78.8 %

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.

2 800 3147 7484 88215 9835 8318 4208 96012 34710 89267 10126 90123 42456 21917 95514 710215 24719 46965 51543 49116 720102 4184 961134 702397 3634 22921 7353 9074 0943 2512 077 2795 2303 3053 8575 1904 2606 069262 781956 05011 52825 14323 22514 8515 98046 93210 09313 616201 68722 83322 31026 68642 79218 68953 34339 69756 76494 554118229 9581898795 764 5265215311416024547277 8590510152025303540455055606570Phred quality score10M20M30M40M50M60M70M80M90M# 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