European Genome-Phenome Archive

File Quality

File InformationEGAF00000115735

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.

4 032 8018 501 25920 120 77743 449 34581 847 470134 426 138195 003 593252 864 599296 414 757316 983 306312 240 646285 152 731243 085 857194 443 920146 778 619104 947 95471 411 31246 447 72029 085 06217 592 20810 390 3036 072 0503 568 4472 173 0871 400 337972 740736 074595 719502 474438 558388 028350 106317 075289 331266 264244 499225 249207 696193 605178 050163 273152 194141 352131 335122 789115 867108 992102 74496 49089 37583 34478 63874 49670 37066 64562 85460 06457 24855 13952 89550 21848 00646 32744 67942 14840 44638 79737 14135 36034 28932 78630 87629 22328 53427 16026 24725 38224 23123 95823 11422 16921 11019 94219 59418 73118 34517 47217 07316 19015 53715 04114 34514 28113 85513 56613 00312 63312 44512 02911 42411 06210 96910 31910 13810 0579 6599 1468 7418 5358 2827 9677 6427 5347 3407 1036 8746 6086 5306 0705 9455 8455 5305 5825 4355 3615 1424 8014 6374 4584 2774 2574 2503 9693 8263 5963 4813 6013 4093 4333 2993 4153 1503 0903 1312 9793 1813 0953 0803 1483 0602 8102 7862 6722 6352 6712 5982 5912 6272 4922 4132 2272 1502 1182 0241 9712 0361 9571 9641 9781 9361 8741 9551 9091 9121 9321 8111 7591 8381 7491 6601 6701 6831 6011 6031 5721 5671 5991 5071 5481 5431 4861 5311 4571 4811 5161 4681 4351 4431 4041 3721 3711 4231 3641 2961 3681 3271 3291 3331 2511 3521 2941 2831 1851 1581 1131 1211 0791 1061 0681 0461 0061 1111 0181 0801 0611 0309711 0079531 0331 07595896292793094697898089989694496291388892692088886990490187385786087576575478775881272973875873677677383773078178576967966771071369260663369871469170166561362262764958955857859260557254954258854550658257653158755552954857453155155552447349056251648553649352548748746449551452348247251349952548650546044449949350547549349546244045241143142541344643044539844141041143842842542238441540442135435737333334441336635434438834638737938236537839037634532640236436633833333334838236236637035232435233136537229934334933326331533230431634332930735331034533333630233535729634130832029229533236230230829032028333028230627926426225623725526825824627927529125927927021725625725824827326725427424125626025822823824622624724926325523426327125223222426425825424525423624624325421024623626221124623722122022823522124720620828022724623521521621523719522318920920919219320922020021124622524125818925322121219621622023422121319621720918922721020620219518221118520122621220621416018418718420020219921618318118717618318417720921118619617519822121520423320917719319819418720920120922621321123019921022619222722722119220620419524917819718518020419617319918319119120219821618821017717216919318619219817419718315717417417917515517517115716817414116319615818814617118218415417421018119922919719315017016018518917718316719415616314217217918516818617817817516416417117715118119314815316015517015516218116117414816415416918314214015416713415915114813814814214413914714315013916415514514413814414813715014315411916413316016216114614915716416011814414914216513915815614414714514113614813817514512614715113514214115314815915213814212915515117615014414511615411512015111211813414414516414013213513714814213212612711213911611514010713012512011112414013910412713712613012711811011413213613613414611215914213615513612711511113213511913314813412512213111111211712212011814214411510413014612413812610112198102125111104121127115105146105116110107115117109144146109144137137125129115140133136111111130122117120106120133121113124117133981271161131331161001101151151001211059894122111122961049489851091001131099910110110793979280100931051401031159712791107114939111111293829192105118103918591961129781107918195751009610083891049390106881169683901021029880102118105849485948910598781086392988510879137 021100200300400500600700800900>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.

2 739 169359 114766 0465 243 81512 085 73110 224 00497 754 98742 358 01812 156 211128 727 270231 167 273166 732 30230 833 01433 519 11615 402 37437 032 08638 578 295132 409 321141 028 743216 899 821157 965 775239 592 989160 516 512133 812 678125 607 558311 199 888249 711 405175 443 166164 649 554301 502 812334 065 104401 634 435560 747 659414 512 780861 891 2871 336 837 8681 019 143 7641 897 834 4102 310 436 8803 465 989 7653 092 087 4823 837 774 2594 273 740 9153 829 190 8481 297 896 243610 927 153291 867 844109 483 63915 292 33136 597 63019 790 657005101520253035404550Phred quality score0G0.5G1G1.5G2G2.5G3G3.5G4G# 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).

95.5 %318 846 92995.5 %4.5 %

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.

94.9 %316 873 26494.9 %5.1 %

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%).

0.6 %1 973 6650.6 %99.4 %

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 %166 968 81050 %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.

94.3 %314 849 60094.3 %5.7 %

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.

1.1 %3 618 1661.1 %98.9 %

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.

24 539 97284 79240 227146 02442 98067 14655 23679 22664 233506 958232 975185 497442 096111 580104 207999 140127 5501 214 037283 61379 430452 87024 631251 7161 161 83915 954275 00116 20616 67816 58518 287 67522 33116 50418 32822 80219 14826 494712 5584 606 45755 94832 64486 88872 82436 502142 95251 30468 056410 57690 694106 490107 010202 21472 842204 604154 282220 650340 636677 362275 432 446051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M180M200M220M240M260M# 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.

99.38%99.25%99.64%99.48%99.6%99.67%99.49%99.56%98.72%98.73%99.17%99.64%99.69%99.64%99.51%99.18%99.08%99.42%99.13%99.59%99.36%99.46%92.4%99.49%0.62%0.75%0.36%0.52%0.4%0.33%0.51%0.44%1.28%1.27%0.83%0.36%0.31%0.36%0.49%0.82%0.92%0.58%0.87%0.41%0.64%0.54%7.6%0.51%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped