European Genome-Phenome Archive

File Quality

File InformationEGAF00004837734

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.

158 649 856254 441 205321 117 542353 069 376347 267 139315 502 705268 270 091216 928 904167 754 617125 328 72990 841 10464 275 37144 574 57430 407 24220 514 47513 712 1569 127 8546 095 7714 075 8042 754 5551 890 7971 324 488953 232706 035542 698425 673346 677289 661247 384216 783190 033165 545147 887133 965120 816110 727101 95394 13386 10079 62672 78067 60862 77657 40354 81050 75448 96945 11643 11640 89438 25937 23534 67234 16732 84331 31129 43628 35026 66025 94924 77023 64623 10922 09921 48120 46920 41519 28019 08618 61918 03917 77816 62716 70215 78515 62615 02614 69114 56613 43013 32312 75612 50312 17912 02911 47411 38511 04111 08510 70410 43910 35710 1219 5089 7669 9119 4559 4658 9128 9048 5938 3838 3808 2388 2717 9097 6107 6947 5307 3086 9227 2817 2907 0867 3217 0916 6756 7656 3296 1316 1796 2316 1195 9216 0765 9175 7105 9275 5205 4565 6155 3685 2975 2275 3035 1685 0295 0885 1494 8144 9954 4684 4794 5434 4154 2884 4164 3484 1954 0934 1254 2734 0724 1874 1554 1134 0074 0123 8553 9773 9493 8433 8823 7783 6423 5003 5323 5753 5103 6303 6173 4133 3313 2293 1693 1713 2283 2573 0183 0153 0092 9382 9832 9802 9043 0242 7572 7232 7372 5842 5262 5842 6682 6862 7572 6572 5672 7122 7062 6472 5872 5922 5812 4392 5622 4192 5472 4062 3302 3132 3552 3892 2462 3762 2212 2402 2832 2352 1152 0922 1812 1432 0482 0972 0572 0671 9731 9552 0011 9531 9311 8421 8931 9241 8571 8261 8531 8721 8871 8241 8091 7591 8951 7861 7351 6181 6741 7031 6891 6901 6251 7171 4921 6281 6011 6731 5371 5831 6501 5991 5781 5001 5601 6181 5651 5991 6091 5431 5611 5251 4921 4311 4651 4261 4281 3791 3951 3631 3991 4051 3701 4121 3841 3471 3081 4871 3811 3901 3681 3051 3601 2901 2721 3291 3081 2931 2741 3081 2601 1891 1341 1551 1861 1861 2141 2451 2301 1721 1481 2401 1891 1091 1351 1921 1901 2191 0991 2421 2211 1751 1311 2261 2191 1561 1281 1131 1051 0881 1011 1991 0881 0371 1391 0801 0661 0161 0531 035993948971957976942930971998939931914954857912955870962939939902883922947910897876963875935993990861837897794873832836847832869785805784832816737722779754773837763804725708711712690755721719780708704721758785794809748743793780773822802843784732738724732671686724798733773773734708778738713778740731777776808730793769785822735754779772833776809713741755710694705757725738790772747702728770718683682706755755727764737733661744683734770720702737771710755731740698725714712725695720718702686673696684739725689726718769756701751681691680697691684729709663661665680689719613688652647640649648704605633658652624632598660633538603607604596629594634565611614564625630581532607557617559615528639634581515591529559618590582565546601549561529540509558565607529560551530528535509536477511502592508540517507499523449497469453495498477477478456504533453498493502481459458487470468446454433428469468424448440418430412420432400420396445479402434407449412449480452444410404422435400433411425420400372396406405411406387350407349384364387343370328386365363356366354346386372394390342402369376420401394352394346442354347384382355381352346340346338358335371350331318376395323332339355342322356289357329323289270302298307298297313325311297297316296324295300300254279320259285310303329288297298284263298289301297293282298292287273314289284299279283281268283294272323289285244281274288266268274264267271278271283292268262259243272276242267255224256288257288328296300297261250259268266276230268258287259277235233260243279275325268268260275303247283252275262267237263257242244285286266261239287240254230243242224239242233221251218218259271255259237286252246289232221235220227255282251250264249229249243246270229246247263249255254232239257285244287236285270272271255287266232247243230263268236245276264245265253242230230232256226239227244229230214230236227218235263206210259232236232247259245241270203246245228242235230228234212260230258223218234238230246246285271300235246272260256257234265232235258237239259228229209 865100200300400500600700800900>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.

1 145 870000000060 678 987000975 380 240000000000564 362 5030000562 832 92700001 173 264 67000002 340 721 64800011 800 551 11100510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G# 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 %114 551 20199 %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.

98.7 %114 287 92498.7 %1.3 %

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%).

0.2 %263 2770.2 %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 %57 877 27850 %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.

94.2 %109 069 04494.2 %5.8 %

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.

16.5 %19 083 24716.5 %83.5 %

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.

7 135 750104 42071 775128 021100 859109 015119 396157 53493 938123 58966 05354 92066 85872 46840 57283 06760 76767 14684 825117 110121 751127 926171 201131 284201 761308 01430 720520 28338 33634 82658 24964 71741 75979 10336 88037 77651 71464 63425 76199 0771 151 25776 75481 880126 630110 869192 620163 081276 705357 31063 28768 50163 93274 44045 829102 26275 18260 734152 71165 949114 887105 512 818051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M# 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.

99.78%99.73%99.77%99.77%99.77%99.77%99.78%99.77%99.78%99.78%99.78%99.78%99.77%99.78%99.78%99.8%99.79%99.77%99.81%99.79%99.78%99.76%99.82%99.71%0.22%0.27%0.23%0.23%0.23%0.23%0.22%0.23%0.22%0.22%0.22%0.22%0.23%0.22%0.22%0.2%0.21%0.23%0.19%0.21%0.22%0.24%0.18%0.29%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped