European Genome-Phenome Archive

File Quality

File InformationEGAF00002386404

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.

142 951 274242 583 940335 144 639393 033 274401 449 729364 699 679299 750 133225 910 026158 111 979104 006 51364 930 49738 840 58522 495 71112 788 4867 253 6994 135 3822 458 1521 546 7141 047 994760 179594 244487 640416 023356 464314 919282 865252 755230 959209 114189 755170 561155 522142 956130 599122 034112 000102 45495 13287 84582 14576 13670 56366 16262 04858 43955 04853 21450 08548 23545 38442 92841 88339 56436 72036 29334 68933 60032 41730 96829 12427 89027 16326 73025 72025 21424 27523 58322 20521 73621 38920 67021 26720 06019 49618 62017 83917 56317 23316 97616 45916 37815 83215 56914 88914 44414 20513 79314 07613 40713 14612 92912 78312 51812 29011 89011 59811 60911 26111 40610 93810 64110 40110 50410 43810 37610 0149 9689 3189 4589 5599 2159 3438 6618 6498 7028 5388 1328 1208 1448 1178 1097 6417 7237 5267 3967 3637 1356 8326 7956 7926 4096 3596 1816 3116 4896 3256 1536 0095 9026 0245 7085 6935 6105 5115 4065 4305 3225 3235 2415 3275 4375 0945 1175 0354 8455 0104 9465 0045 0454 8664 7244 7054 5994 5754 4854 1864 3204 2834 1893 9563 9693 9543 8703 8393 8473 7533 6853 6873 7113 7103 5893 6253 6103 5713 4293 4763 2333 2933 3623 3033 2693 2503 2653 3393 1933 1443 2013 1293 2563 0503 0862 9962 9772 8002 9562 8162 9692 9552 8682 9252 8252 7522 8662 7552 6322 7442 7102 7402 6752 6482 4442 3452 4442 3802 4682 4472 3572 4282 4692 3502 3472 2542 3992 4392 2572 3002 2562 1662 1062 3262 2862 2862 1662 0802 1892 2082 0082 0411 9581 8961 9962 2391 9702 0781 9631 8781 9211 8491 8781 8721 9521 9751 7581 7871 7961 7161 7111 7091 7171 6971 7391 6231 6501 6241 6171 6251 6961 6691 6341 6161 5231 6181 5751 5881 4831 5841 6291 5821 4341 4531 5201 5231 4111 4681 5281 4501 4791 5541 4351 4711 4691 5191 4061 3671 3521 3671 3761 3581 3571 3201 3361 3131 2961 3891 3431 3011 3371 3421 3271 2721 2841 2411 2721 2151 2561 2621 3231 2711 1461 1301 2071 1891 2491 1841 2481 2091 2011 2231 2551 1621 2111 1791 0691 2281 1541 1101 0761 1251 1271 1291 0661 1571 0651 0621 0801 0281 0581 0391 0301 0021 0291 0099389819789951 0039839319379959969819101 0149719379709379349549208738671 026896849922983906959991947947896963920900902843873877856828813817842757781862758772785794743791753740785756748725714709738764713773765732765731742885723710684668683692769723731750811763820719778671661654694682721725690724699657704695687708706698653690663665678694688734676691652662631579637692637683640707641680630653673575647653689601649634654601601567607635645578582603573584598541628555609556583608529578580599595607609608580593577605612596571563565594590559601553590594613654571585599557580584570571546597597604526592592564570592525524501555552528553547545527539530429524511493500511508464531514528550530509531511496530527549522542507557549574531536572504573546527548570514498558507532605571608605561581559552550582588519554548518530519528515500533486455479524469490527521473499579510538543545509539520509527587530511513505575558532615515577565509638609583543553557544587591549581547573576552533523518505522525574511512525547491484522522545534516548489514506493500536624524492526489500483479479473491528426467500459419421432436403410410413413393469485471457408438430401379401433402380415426420400419417384373407399393399405423450418407422406392394381406388393336340373345319321328342294327358326365344333359329314314291309339296303314279224300284316286309296290272285260276281257287280307254260264289257249260278286284284253251237263238282254237203221275234246277258231236205193233218236222225204204220217199210203224208194213212197192195210212224203211217225204209180200231197215225208189207191179197192213203191188187174184196206195185175171173189190181176191163214171152162179193175183196160181300168172179157192177166190159158180184190170169179177153167193198153155177180214208190261177204223185199174191166183171177163183164152192152166153164191167183193146174160175177192162172175183172187164176179166171181206194166167178161181175145178210 901100200300400500600700800900>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.

1 850 9980000000230 626 7070001 716 376 197000000000912 931 1780000917 926 53500001 483 478 89700002 591 421 8200009 039 950 49000510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G# 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).

99.3 %111 065 68599.3 %0.7 %

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.

98.6 %110 289 29698.6 %1.4 %

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%).

0.7 %776 3890.7 %99.3 %

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 %55 942 26150 %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.

97.3 %108 830 25297.3 %2.7 %

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.

9.4 %10 526 5499.4 %90.6 %

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.

5 753 843118 74972 591135 344100 364102 535134 884156 67667 585121 75956 10350 42184 57081 96843 41093 93668 85480 736118 988146 609147 074132 953166 090131 526217 107337 98020 520557 63230 32829 88968 01361 43125 99774 98730 41730 96762 61267 67017 95298 0841 725 37977 99389 252118 193112 012190 483159 529260 725292 44259 67963 79564 89571 87252 21785 38685 41372 923170 57781 163121 96498 430 970051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M# 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.

99.27%99.27%99.28%99.29%99.28%99.28%99.29%99.29%99.27%99.27%99.26%99.28%99.28%99.28%99.27%99.33%99.27%99.26%99.31%99.24%99.29%99.29%99.52%99.11%0.73%0.73%0.72%0.71%0.72%0.72%0.71%0.71%0.73%0.73%0.74%0.72%0.72%0.72%0.73%0.67%0.73%0.74%0.69%0.76%0.71%0.71%0.48%0.89%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped