European Genome-Phenome Archive

File Quality

File InformationEGAF00008487545

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.

19 695 66716 916 67010 002 1468 891 6514 092 9354 385 8821 911 1862 446 9191 149 1731 447 096782 575922 385566 461633 469423 198455 472335 055345 489260 153268 834207 670212 917174 860176 680139 882147 821121 445129 690104 277108 38792 38698 72982 29288 71773 89276 99465 53571 28560 44163 09656 58356 75050 83851 15346 25447 71743 32143 01538 83640 51637 03136 58134 50333 09631 56532 06430 59229 96928 42327 47226 20326 21524 82924 40623 47022 91621 74221 24620 52320 17620 42719 83018 96818 75119 04918 45717 51317 62217 26316 83916 37115 72615 55515 54115 09214 66413 21013 25212 89312 89612 89712 30012 38312 19312 09611 56911 29811 31710 77910 44210 2709 9839 8339 8519 3159 3449 5219 1229 3148 9998 6698 7638 3928 2857 7117 6467 6587 1807 6156 9986 9096 9196 8616 7586 7826 5946 5876 7536 2676 4356 1546 1765 9175 6965 7175 3885 1815 4045 5655 3405 0085 2195 0784 8994 9444 7894 6864 5634 6624 3924 3494 3504 4544 3464 1464 1664 1444 2354 0143 8833 6793 7253 7773 6443 6213 7873 6253 6083 4993 3993 5693 1893 3973 1893 1263 1473 0543 2583 1972 9243 0172 9942 9982 9642 6892 7952 7142 7072 7752 7772 7282 7572 7392 4802 4902 5482 4492 5522 3932 2382 4832 3622 3132 2272 2302 3792 1002 2232 1192 0622 1392 0931 9501 8852 0671 9561 8372 0092 1351 8861 9241 8292 0872 0111 9231 8661 7801 8581 7961 7421 8441 8051 8461 6591 7151 6951 6141 6671 6311 6291 5601 5741 5541 4531 4951 5611 5101 6331 5261 5171 5121 3341 3301 3831 3911 2591 2941 4031 3441 3901 3171 3461 1641 2461 3161 2771 2731 2131 2641 2241 1891 1231 1651 2071 1261 1681 1301 1661 0631 0529431 0281 0451 0291 0999991 0451 0131 0189379188951 0459539778598289059659128188798328878198808459568029028818948629229498779278699458057818978657868437417597638916807327257377377376967336337266637866627246717757265966386776666896246776816415876885926056126236346565776316195875585716025696345555015286094905774904705175285685235464885065165355245094554384684864605024834824804713945104535604904714774594814104414214154294503864113824334244714223934373873994233983864113583994353573774134314273994444053763893443763604612963843454143393343663493593313783483743633973563383323823722902983023693813182772843462843143503583622682733402703142872893002952853313132983042712672703332872652782552553203172833243233072652502382602982682552992502732501912352712742332052672322002792602452752592761872982842642352272713132352672832312622052492432441942582472592402732142492102311942612342632562392322202102292432622892792331722472102752312182352232191992002022312142342582332291672152082132521872202152342462561942252181832282102332011921961882082082271682121872021881792291821862081571911881771822411702121652001801662021671931661541802232001931972101921801631901721602041701761871941991741611771671521491571861941871761791881901621781742191561771811662291861571771581681831201671711851781911631961821361881511481741701811821581981591901621791481591961401371511771731641821501791631891821391711801781881711741551861781571721671711511551401581941601451391601731561741301561581291671601531711681671531591251791551541431721421581291591471471641591051471501541541261421471461601321481291321251491551401651691491721661561381431621461331131001061251461241171671471241191491561521352051381711701521251621781601441301731602101311481501291381681131471331221371851351301361441251121461351491431241321091371121331741471491451381761451281391381141311371481351351751161311341561371311211141271401341291501231241151641151201531351391311351451151581171351571041171301351051261291241111241271181271561421291301491491701201171231131251461281591371441281121421211339913110397108132139991271341031221271301331161221421141061161001231011551251031141401159711013311213212314411910898101136143137129137544 813100200300400500600700800900>1000Coverage value1002001k2k10k20k100k200k1M2M10M# 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.

00105 93500000215 88600071 760 8525 430 16114 702 61618 821 92515 790 06402 135 175001 053 95353 652 9012 636 6563 865 7947 521 0873 751 79863 266 5446 706 62610 935 7752 005 22413 597 429136 830 80032 074 05033 120 65720 392 29233 741 831294 197 030792 728 577001 375 846 86200510152025303540Phred quality score0G0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G1.2G1.3G# 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).

99.9 %24 106 88699.9 %0.1 %

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.

99.8 %24 084 26699.8 %0.2 %

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 %22 6200.1 %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 %12 067 55450 %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.

97.2 %23 463 86297.2 %2.8 %

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.

87.4 %21 104 29787.4 %12.6 %

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.

437 08316 96410 88922 90712 13513 55015 80917 52411 58422 01210 4909 32910 82011 0245 16418 9277 1499 58512 09811 5787 47217 4769 81311 08815 10024 7476 97587 3227 3777 99310 55212 4556 66719 3567 7548 35810 08111 6856 30728 646225 43813 61915 72814 25618 42526 93631 35033 32154 253103 65213 47323 88814 75537 95012 53212 75213 44437 57320 82230 01922 598 861051015202530354045505560Phred quality score2M4M6M8M10M12M14M16M18M20M22M# 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.

99.92%99.9%99.89%99.91%99.91%99.92%99.92%99.92%99.92%99.91%99.87%99.9%99.93%99.91%99.91%99.91%99.91%99.9%99.89%99.9%99.88%99.91%99.58%100%0.08%0.1%0.11%0.09%0.09%0.08%0.08%0.08%0.08%0.09%0.13%0.1%0.07%0.09%0.09%0.09%0.09%0.1%0.11%0.1%0.12%0.09%0.42%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped