European Genome-Phenome Archive

File Quality

File InformationEGAF00000487831

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.

95 422 80222 678 91810 275 2806 306 0314 511 9223 505 6582 874 8662 427 9722 106 5421 854 0221 644 5951 481 3851 340 2271 228 2791 121 8151 037 514961 754897 616835 176774 671724 445680 331635 855599 824563 480530 031500 035476 559451 606428 323407 523388 352366 976349 917332 775318 655304 910288 865276 658262 934253 494241 360230 578222 756212 299202 081194 548186 393178 481171 369163 980157 666151 947144 390139 165134 228129 886125 204120 290115 831112 233109 116106 327102 63099 72596 76194 10190 93389 14786 69184 24580 98579 42576 58774 56072 52370 79167 93266 37864 76063 12061 06961 30158 15757 17955 14954 29553 25751 90550 15449 14148 23447 21145 49844 09643 27642 50641 02040 71239 56538 79237 94037 52936 21035 04834 35933 70233 13332 78232 37131 37631 03629 94629 16829 06828 27927 32127 19925 99425 15625 04125 16324 05323 62723 08722 37621 76921 78421 18321 09220 67820 40919 99619 76419 17318 69919 09218 68018 15418 08817 74117 23317 34016 83016 58016 34215 94615 76815 56115 68515 50615 11515 04314 54414 56914 07813 82013 90713 81813 31012 91712 85712 84512 48212 30512 16112 01411 60111 51911 54911 11311 46610 90110 90410 77310 40610 83910 32410 37310 01710 1199 9409 8989 5459 6099 3619 3849 3389 0739 1958 9328 6998 6078 4398 2328 3268 2818 0268 1127 9027 6217 6947 8177 5557 5187 5487 2697 2307 1576 9927 0336 7706 6926 6496 5036 3656 4426 2406 2506 1786 1686 0036 0835 7855 7835 9395 4585 5255 5605 4825 4385 4225 2885 3205 0945 2345 0455 0935 2015 0194 9734 7824 9574 8484 9434 7974 5714 6454 5614 4834 6404 4734 3444 4544 3624 5624 4274 4204 1384 1894 2964 1144 2134 2684 3023 9223 9924 0124 0143 8513 9133 7783 7823 6883 6783 6753 7213 4683 5083 6473 5133 5583 5453 5543 3933 3973 4003 4313 4193 3063 2853 1983 3073 1713 3313 1663 1783 2963 1083 1632 9882 9942 8753 1103 1123 1303 0552 9772 9892 8212 9102 8052 8502 7222 8632 7192 7722 6472 7422 6932 6912 6822 6062 6542 5612 5742 5832 6432 6452 5582 5192 5912 5032 4502 4692 4032 5042 4102 4602 2872 2532 5152 2842 1362 2012 2452 2532 1332 1672 1592 1822 1442 1792 1562 1012 0902 0452 0851 9571 9491 9252 0351 9651 9212 0482 0471 8751 8461 9281 9431 8821 8471 8071 9021 9571 9311 8871 7671 8721 8151 8971 8041 7011 7131 8151 6841 7971 6741 6091 6121 6191 6441 6171 7031 5791 6521 4701 5661 5241 6131 6531 4801 5651 5471 5121 5551 5651 4801 4641 5041 5041 4351 5611 4131 4651 4571 4641 4121 4631 4451 3691 4061 4121 3771 2761 3181 2441 3191 2991 3121 2511 3581 2651 2651 2511 1981 2621 2471 3181 2041 2361 2191 1931 1531 1761 1761 1811 1441 0701 1411 0841 1271 0991 0491 1291 0601 0721 1121 1861 0941 0821 0641 0391 0781 1111 0951 0431 0771 0529959461 0299781 0281 0689851 0011 0871 1331 0529889999879349111 0161 012972891981882884891904968919891859893876900853825850882863821784811726802790779789787851744784763773794741788734691703748714756698696711739744678690726716761732658683757674694650668589643615633671654706653627609573642608608650672658645578586620628579649567583606619599637631615613525560595682651575577581600596548530522581562592582586579507505510516543540610568521550530547512512480558564513476454484539468487469483514545488464474459466453522528498467485433497474452461455482449493447496483467430470472499471429440424437447452453446413502457433435431416393390440496386405418447433405411440403405421403392419451407415427390385390399373411391367376376372396377398367349348346343317367330345352328318365365351344340368339343352359336323277296324312303334296329301332359334326312314326310326324360332294321305290279293293280283299294305304334275296316315324314302313309317328317288328297283309299297266311278316356321301290293293283304316265306273332259300306294301306311271276291300263272268266288245288256260283274242266266295302292267273228234267260280248277268253271263263293230257264290267272239252245252255272270229207230264215225205216244238211234225239250224221218229238215265257262213249239209235224224238265239235231252227209217201229217245225238224229222207209238210226214225213229203228237232201208191186180225213206226193194222176186162217188193201203189184189191191205209240199199184200203205205200173187157202186210185195178209202182168182175178167176178178176217175169152156218174216156165178168200192203177186175148170174167175148 530100200300400500600700800900>1000Coverage value1k10k100k1M10M# 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.

125 59700003 011 3551 134 9214 887 9183 839 001860 8391 922 004822 8691 292 4921 349 092646 0252 972 4321 252 0931 635 1763 134 6401 460 5704 921 3591 958 4933 667 0995 377 6214 833 0035 081 5649 164 1068 454 2887 333 5529 780 78318 498 52236 590 55120 535 18953 168 919127 857 430220 095 14475 771 281258 527 099124 914 581311 537 971330 101 945892 998 27600510152025303540Phred quality score0M100M200M300M400M500M600M700M800M# 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).

88.3 %30 159 51488.3 %11.7 %

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.

83.2 %28 402 15683.2 %16.8 %

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%).

5.8 %1 757 3585.8 %94.2 %

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 %17 076 77250 %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.

73.9 %25 253 92073.9 %26.1 %

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.

14.5 %4 960 05014.5 %85.5 %

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.

7 641 921171 2886 75132 78814 45611 95813 80312 48710 833229 70525 283331 36497 92314 91640 626274 33923 281171 95993 26624 513187 4214 20686 012560 4852 276158 5672 6722 1622 3073 885 8223 0822 5742 4063 1022 4763 466253 1084 917 4858 4702 69012 21212 1902 19446 1903 7305 616194 9585 98612 0167 14015 0545 67815 23811 25215 91839 856152 86014 261 207051015202530354045505560Phred quality score2M4M6M8M10M12M14M# 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