European Genome-Phenome Archive

File Quality

File InformationEGAF00002240866

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.

5 428 08110 072 46717 848 19129 311 44744 744 23564 719 06489 681 923118 824 824150 413 023181 302 464207 855 068226 594 649235 344 519233 440 227221 859 672202 455 456177 991 682151 194 489124 526 40899 452 65577 357 49258 659 34843 578 90931 744 51822 752 71516 083 33911 279 6247 861 5795 485 1773 854 7082 752 4592 000 9831 501 5381 159 198932 035777 026665 368579 640515 688468 789425 265395 320368 796341 518318 433298 447279 394262 059248 693235 924223 392212 138202 068192 278183 251173 808165 741158 825151 674145 184137 930131 570126 740121 057117 134112 475107 414103 82899 95096 97792 47088 57086 68484 10981 84777 96175 80072 35568 51666 58564 38261 98958 53257 32254 31951 66649 47347 67045 51043 79342 66541 36440 09439 21037 47536 23235 58534 05533 26131 76830 59229 70729 08727 84027 08225 89624 91124 42923 76323 27221 88121 29521 48121 00220 60219 74419 32318 90118 50117 66217 34816 98916 88216 40415 87415 33915 08715 23414 71514 97214 11913 97013 75413 53013 50312 95412 57612 50412 45711 92311 80311 56311 49911 18011 10411 16110 65810 59710 52510 42810 45510 26210 03410 0379 87610 0079 7949 4909 5319 0659 2969 0949 0489 0958 9958 8708 8128 3628 3298 2778 0788 0137 7788 0187 8517 7527 5067 6497 5887 4797 3297 1117 2267 1646 8626 9246 9646 8686 9396 9697 0126 9866 5736 5626 4846 4316 4006 2136 0476 0545 8756 2925 9885 9905 7905 9315 8325 7565 7505 8095 7755 6335 6715 5975 5245 3525 1895 3235 2485 1735 0604 9644 9834 9304 8494 8624 9964 8214 8755 0194 9474 8704 7354 8034 9164 7584 8424 6194 5774 6324 6234 4054 4374 2174 1544 3264 3934 3144 2744 3324 3294 4974 3604 2364 2954 2244 0594 2654 0444 1584 0524 0743 9723 8403 8183 8743 8433 8833 9093 7013 7673 6183 5663 6693 6603 7133 6173 5943 4893 6363 5933 4573 5143 6303 3313 3473 4053 3263 3103 2383 2943 2213 3293 1723 1243 1413 1613 1032 9582 8882 8982 8432 8982 8532 8972 8242 7882 8012 8522 8822 7312 8542 7902 7702 7232 6642 7262 7432 8622 7552 8202 7842 7532 7512 7012 7092 5942 6372 6852 7252 6402 5982 4612 4512 5042 5442 4992 5852 5832 3932 5562 5162 4342 4522 4712 4222 4492 3302 3382 2912 3482 2302 1492 1262 1482 1042 1562 1842 1362 1182 1472 1042 0792 0652 0982 0932 0382 0342 0442 0631 9951 9761 9802 0561 9721 8992 0381 8961 9441 9361 8661 9451 9331 9021 8811 8071 8361 8781 8631 8931 9211 9491 9401 7921 8381 7411 8141 8341 7671 8521 8401 7741 7191 6891 7461 7971 7821 8231 7021 7791 7991 7211 7501 6961 6741 7711 7011 7441 6921 7001 7671 6521 7101 7851 6701 6781 6261 6551 6411 5731 5581 6251 6211 6331 6301 6161 4891 5581 4851 5311 5761 5361 5221 4921 4511 5121 5121 5121 4161 5541 4661 4931 3601 4091 3661 3781 3601 3901 3121 3451 4071 3411 3861 3611 3781 2931 2651 3451 3751 3131 3121 2861 2681 3221 2801 3511 2801 2901 3061 3521 2591 2091 2261 2721 2121 1911 2311 2181 2811 1891 2391 1861 2211 2001 2541 2681 2111 2081 1891 1651 2011 1891 1781 1791 2261 1501 1951 2081 1861 2191 1701 1801 2031 1811 1391 1271 1681 1091 1731 1761 1401 1031 1021 1771 0841 1331 2041 1441 1411 1161 1531 1381 1031 1771 1961 1971 0711 1301 0831 0551 0761 0111 0691 1031 0651 1021 0889921 0121 0831 0341 0471 0701 0521 0801 0081 0621 0201 0201 0171 0331 0491 0731 0221 0151 0181 0001 0161 0499981 0421 0389811 0249811 0229979811 009972964975958956974933977913955898927920880911973918997939917897912891915897829874853870850921875884908874876894873859876839797820793794827763838764803829798788792802833845879831901836832798812829846795818794753801805791784838776851831833819820826821822852787820841838736754765775766737751696754733746754726762737751754758719795737744721813753741761726756752784699714709740696687697654657699690714664669712665675708633711660674653686664661707681666645692683664649679679669690665718667703690654663687657648595629596621624627626638638634627630667675634651667655639649631585604617638601609608615582660642663610618693649627638568642638622606598625572659579619593563619607539552589552581579609571578593592534583538557540551503554558504588557536550556530544557559541568539518515550514531550553540536512510577547562531569537572475595520501483528527500495513502521489484483504510469485514498476473495474476461469496459464477480492503472509525547505526445444506505496534479520435474485455452470459440440454457439498432442383409436430461461448439396420424438382415448415429420448428423463392471449443463431432392398402419402411361362411386376393352402375324404361402379367350355353356382388370383383420406378340376375370407376373419357375337397371346371393335378368449356367512 634100200300400500600700800900>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.

5 877 418000000081 339 8170001 566 949 410000000000920 372 18200001 076 126 66900002 374 786 93700004 951 098 68600032 437 009 50500510152025303540Phred quality score0G5G10G15G20G25G30G# 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).

99.6 %286 299 35699.6 %0.4 %

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.

99.4 %285 813 26899.4 %0.6 %

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 %486 0880.2 %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 %143 753 51250 %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.

97.9 %281 551 74297.9 %2.1 %

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.

10.7 %30 825 65210.7 %89.3 %

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.

13 218 805273 333162 016326 442243 287256 596276 464388 344166 185298 834138 026119 138165 149191 52095 977229 906168 113185 608230 362320 599320 386327 104429 131333 368524 947828 69848 5361 439 25076 39373 924143 926155 27468 334187 24378 05276 266122 880164 78042 398244 1203 583 201169 431161 082280 722237 209442 869377 371585 988939 036103 031136 981119 064154 07868 372125 893136 59796 708383 611101 712213 023257 367 682051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M180M200M220M240M# 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.

99.83%99.81%99.82%99.82%99.82%99.82%99.83%99.82%99.82%99.83%99.83%99.83%99.82%99.83%99.83%99.84%99.84%99.82%99.86%99.83%99.82%99.82%99.87%99.86%0.17%0.19%0.18%0.18%0.18%0.18%0.17%0.18%0.18%0.17%0.17%0.17%0.18%0.17%0.17%0.16%0.16%0.18%0.14%0.17%0.18%0.18%0.13%0.14%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped