European Genome-Phenome Archive

File Quality

File InformationEGAF00006165002

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.

321 087 359465 131 100510 913 606463 100 549363 484 404254 152 192162 282 59596 226 45253 721 82328 646 05414 839 5127 668 8004 032 9672 222 8741 328 288868 876620 417486 304397 784331 590285 969242 995208 127182 938159 808141 303125 559110 60299 19388 42179 70573 06967 12461 16955 84552 61049 66446 10743 56840 98538 77237 09135 41533 57432 14430 21628 80427 28926 33725 36124 87623 96723 69022 22521 33619 37419 39319 15318 61017 91217 27816 78215 69715 36015 16115 03014 48414 06513 85213 44813 32412 70012 41712 59912 19412 27111 59911 25610 77011 21510 68310 46210 55810 5039 9649 7909 3678 9989 2749 1449 1469 1688 9908 6438 5457 8158 0317 9057 5977 2867 3256 9516 8706 6606 3586 6016 3326 1715 8406 0555 9125 7275 5885 4445 3735 3955 3385 1974 7054 9304 6934 6514 6534 4524 6494 5684 2974 3423 9794 3344 1904 0873 9553 9053 7313 9083 8613 7603 7323 6163 6383 5323 4543 4553 4073 4643 4313 3973 3603 2573 1163 2873 0923 0473 0092 7812 6872 8612 8012 8582 7412 7002 6302 7302 5942 7572 5612 6072 4882 5862 3882 5512 4282 4252 3522 3882 4242 3492 2942 2192 1542 1522 2602 0982 0772 0982 1282 0542 1842 0912 0522 0892 0261 9821 9571 8732 0391 9881 7932 0042 0131 9711 9411 9221 8791 8181 7691 6921 7101 7251 7231 7611 7251 6851 6851 6761 6551 7631 7131 6821 7211 6241 5791 6421 5211 5801 5221 4951 5201 5131 4921 4191 4821 5501 5301 4411 4431 3191 3851 4401 3321 4221 3241 3881 3471 3621 3031 2801 3721 3501 3831 3611 4911 2321 2841 2311 2431 3011 2411 1431 1881 1471 1191 1701 1631 1491 0591 0741 1781 1671 1011 2011 1121 1821 1671 1561 2031 1481 1851 1261 1611 1471 2041 1249799721 0149981 0111 0419989719791 0271 0031 0651 0319899961 0089349219469999819758919499579488979291 0009069341 007955961977948957893863888916867818870842787852789783835879884765864865785832821795776769753838852815776804820891833877771744753840748800798813723755733751820714716718748750743708695707717748696771763774780662741763774772745757730751757759825793719784705658748757699690698647707635684657637647619606609629607625582588557566608564530508565533509547549566522546488515490495521503463479474478516533483538482489556523458506501488493484471491430455493471421431428415454425441463451456435481422422408459424444407452449382417434412443436426420427455425448396467465474444462500442468473482432528454501475432430454477450441455430417399395446407412452421428423478483520386400444466415395386423374373380394370473442403398389375335369327360352333333355320314336334348309345313313331345318286342320328318329321332343311292329310308293314316296286302286297311336271281287317306375382344306329300329290316278311291287263257309299317317286319310319323303297297301284291317270303292284295302264299325287265264271285271294279249271257274252259229261222240265257257231273280245250231264219248255227242215238231233228236204226244259228228246234235217241231225219256221227218220201215184210189197183197201198205183192206191187191204191231193180193198194203193184200183170218191193184198187191178190198308175174191196174171191179169184174183185190186205181182192170189176141168172190142174150176164169188176164191167188178187162172188160173173178166179168189204163201201176192177154183184169160164178151173170174168177171162178177177182161184175192169175171179178190165170195171159172174181213189163165182185184168182180152169170179189177155169196174170171190200178174169181198187198175177167155172192186176190168189170187147181191154181160166174203188205179170164186196174160179178186195188170195184194185181184170166171166183145160175182171163174195175183189180161183152154176168145173180192169162165181153203182168169205169182177180202219193180201180197186183160141203186185184191180180160197183184153167158150173177169173200177193194173215167186175188184168189193191218191156 646100200300400500600700800900>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.

122 5270000000000521 016 9990000000000000725 788 1270000000000010 486 002 47100000510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G# 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).

99.8 %77 574 64099.8 %0.2 %

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.

99.7 %77 495 98699.7 %0.3 %

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%).

0.1 %78 6540.1 %99.9 %

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 %38 850 76250 %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.

98.2 %76 272 45298.2 %1.8 %

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.

10.9 %8 471 88410.9 %89.1 %

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.

3 307 82874 35344 483125 77465 95067 34674 755103 68046 37976 89735 65445 60542 37155 64840 71758 14442 21948 82158 25183 19687 18584 161103 72079 920127 448216 48812 153387 47518 53518 02736 36538 53716 45147 27019 96119 00331 50542 91111 13463 689977 33043 33740 08669 96460 003113 86896 169158 388239 44026 77036 20431 63241 59020 24533 20538 23526 212102 53926 88056 72569 843 548051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M45M50M55M60M65M# 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.

99.9%99.86%99.91%99.91%99.91%99.9%99.9%99.9%99.9%99.9%99.9%99.9%99.91%99.9%99.9%99.9%99.89%99.91%99.88%99.89%99.9%99.89%99.9%99.81%0.1%0.14%0.09%0.09%0.09%0.1%0.1%0.1%0.1%0.1%0.1%0.1%0.09%0.1%0.1%0.1%0.11%0.09%0.12%0.11%0.1%0.11%0.1%0.19%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped