European Genome-Phenome Archive

File Quality

File InformationEGAF00002310230

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.

113 404 644199 743 149278 895 466333 318 566352 860 728339 519 236302 060 295251 762 414198 414 641149 015 706107 545 03674 899 82250 590 89133 348 63521 592 34213 796 3278 746 2735 536 6443 529 6082 302 8181 543 0131 079 920788 781603 278487 790404 757342 865298 906263 217237 907213 086193 638176 979162 566147 760138 167127 161117 731108 202100 51993 16986 94579 91974 94170 02665 09560 80856 63753 76650 12747 07244 75141 96639 43037 61936 13034 14232 00330 66029 74828 44727 41426 12025 40123 78423 58123 11622 34821 31920 08419 63418 95118 98818 79717 99717 32517 03316 71916 12315 52515 00514 14513 91613 42013 17812 87912 62612 13011 61411 09111 09911 22110 29410 25510 44210 03110 0149 1638 9158 9369 1758 9118 5358 4238 5718 1298 1518 4238 0958 0527 8987 7757 6937 5147 5537 3967 3097 0556 9026 9566 6396 9616 8276 5116 5006 4506 4706 0725 8155 8355 8585 5915 5065 6105 6225 1895 2505 1025 1825 1255 0394 9674 7454 7814 7974 9434 9524 7854 7074 7944 5724 6744 5654 3324 2624 1724 2564 0134 0453 9373 8043 9223 8983 7333 6773 7303 6033 5903 6943 4153 6053 5593 5093 3523 3383 4183 4293 2613 3173 4693 4173 3443 0753 2373 2122 9553 0542 8482 8882 8102 7912 7872 7282 7682 7632 6752 7112 6032 6412 6862 4742 4782 4732 4462 5332 4782 4372 4842 2942 2742 3862 2002 3042 3232 3472 2922 2592 2732 2042 2222 2702 2092 2282 2052 2442 1872 1382 0722 0722 1752 1192 0192 1382 0212 0411 9972 0251 9891 9491 9471 9391 9941 8701 8451 8271 8761 8721 8401 7451 7011 7641 6861 7021 6641 7551 6521 6511 6421 6321 7851 6701 7441 5631 5461 5591 6011 5711 5311 5611 5091 6201 5781 5801 6271 6241 5811 5811 6121 5631 5201 5201 4981 5391 4611 4711 4351 4651 5341 5481 4681 4541 4961 4071 4221 3791 3091 3671 3181 3221 2371 3301 2461 2401 3081 2731 2571 2181 2121 2641 1831 1851 1991 2011 1541 1331 1471 1761 0711 0921 0591 0791 0701 1061 0731 0249571 0431 0341 0411 0611 0841 0601 0151 0131 0331 0061 0971 1561 1711 1061 1011 1501 1401 0841 0331 0611 0051 0221 0109561 009966925843873900906879944939936887893847907850922895857887886870910839888876827885826821828848814848823808836772764744789774854833785821842814836819818796833762856822809812821789768793811842870766837857812772748753727762760839832772729731752692718700761767807733765704735665693725729713728671730703752687657626679708657690689697734645673659729711699658670672656757732647653656656722661705676721722686714678673670711681678610693623711660680654723740691697702672682680677698626664674679712632639658619660652723616612629619610623617574646618615599678652703637673632611611642638592562560568614590573552573561523555540583513543537511494537544506489551516525548497526489540522491491503516466523517466472496473456455469485481525427484449483458447413454514450439453458486439458432463429430407390432413403488392429429412443407409408424383435425393402439435399431433416379424415435421410415438432399374415388372414406406373417369369381404413442458380371370360337355349370387371363343361380381374339386330362352360351354395322383388344336346375381342343323347344359354310302310313322358348347354348348318325353336335316310317354329302352375325330388333326335323326322305337307327319351354339317326330348337349353339321334309335314281320286333308291297301299321315294302300309313324273315307322331323306303297309312305314328316284325349335273323322323292307316298293319329288327340309297316270273291290288289272283308305312310313312289281281316243277292301272266271266303265265262275239263227258251245274252252249255258213251249246255240227246235244250247239278247240229230239239234203211224231232236223254197244231234229214234225226237226214249243228208234231223244228229230256237226265230223252241255245239247253236249218220232244228264249224230244226223237255238247231245223235233226235273245237229234223219265203226271245246223231213224274243233255240230268254270264308271256246258265262265265272281279228248280234252276254238288228227249240245233267233272233 606100200300400500600700800900>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.

2 164 557000000031 408 767000807 570 910000000000456 397 9480000521 721 12700001 146 837 61400002 371 959 24600014 075 672 49700510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G12G13G14G# 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).

98 %125 935 89298 %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.

97.7 %125 572 73297.7 %2.3 %

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%).

0.3 %363 1600.3 %99.7 %

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 %64 283 88350 %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.

95.5 %122 729 88495.5 %4.5 %

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.

19.2 %24 639 53819.2 %80.8 %

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.

8 023 229112 53969 492134 833103 041108 577117 599165 42181 404127 17560 69151 68170 36278 80642 71594 68670 44577 00695 422132 385138 575137 910178 441133 115215 290353 99623 451615 53734 12631 22262 19065 07534 28579 88033 30733 59951 84670 66220 256106 5291 464 62074 07969 998122 340100 188187 031165 460244 027424 41047 29762 07554 02171 21632 11566 08562 34944 021168 23645 11094 119114 336 648051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M# 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.

99.72%99.66%99.71%99.71%99.71%99.72%99.71%99.71%99.71%99.71%99.71%99.72%99.71%99.7%99.71%99.72%99.72%99.71%99.73%99.71%99.72%99.69%99.78%99.57%0.28%0.34%0.29%0.29%0.29%0.28%0.29%0.29%0.29%0.29%0.29%0.28%0.29%0.3%0.29%0.28%0.28%0.29%0.27%0.29%0.28%0.31%0.22%0.43%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped