European Genome-Phenome Archive

File Quality

File InformationEGAF00002551496

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.

116 576 982225 393 220330 127 775397 474 128412 027 473377 586 441312 570 283237 310 032167 126 064110 290 15568 812 55940 937 11523 485 57013 136 6217 311 4904 104 7692 397 6211 505 0591 025 378760 580601 725494 885425 678372 358331 343297 479265 641241 466219 121199 593184 165171 757156 675143 624130 956119 930110 587101 40294 76787 66381 30976 56970 86568 13464 46560 66857 31252 58850 70147 20545 14843 90842 24440 35938 13536 06135 31933 59832 56731 02129 89529 10528 29327 53126 99726 71125 24725 21023 95723 65422 82222 47721 42921 74121 39320 28220 05619 84318 52617 93317 48517 41816 80416 31516 09115 66815 66715 60315 29114 94814 94914 28114 35513 77813 42213 09913 14012 85412 64612 07411 78711 44911 40311 32110 96410 59710 37110 26810 26010 12410 1929 9439 7589 8329 8989 5199 2549 0429 0228 9658 9028 6928 6918 7818 5378 3198 2838 0037 6207 4787 3707 5067 4617 5097 1637 3027 0666 9037 0716 8886 5466 6606 6216 1626 0396 0846 1706 0415 9635 9515 8325 7595 8505 8285 6055 4805 7435 3955 5455 3455 1695 1295 0125 0764 9894 9214 6874 8044 8574 7614 8104 6494 6004 5064 3804 3864 2204 3334 3214 4154 3654 3854 1804 1694 0644 0914 1564 0893 9823 8293 7893 8863 7693 6253 9913 9193 6303 5053 5593 6043 5243 5333 5473 4943 5403 3723 2733 3253 3963 4903 4653 2953 3603 2363 1523 0503 1373 1623 1113 1013 0503 1563 1433 1103 0682 9652 9492 8952 9322 8512 9112 8032 8732 8092 6562 7002 7192 6532 7232 7492 7392 5812 7522 4602 4362 5062 4952 4962 4602 4562 3722 4002 3942 3622 2812 3862 3772 4072 2942 2592 2302 2972 3082 2272 2412 1832 0582 2692 1352 1742 1902 1552 1932 0262 1682 0512 0822 0732 0651 9492 0091 8971 8611 8751 8581 8731 8431 8601 7551 7901 7511 7911 7251 7731 7421 7261 7201 7111 6881 6561 6551 5781 6441 7871 7111 5691 5891 5541 6981 6971 6821 6161 6471 5931 6001 5441 5671 5631 5371 4991 5321 5481 5141 4241 5271 5081 4571 4271 4091 4341 3251 2571 2921 2791 3321 3151 3091 3981 3041 3401 2801 2011 3011 3001 2751 2341 2501 2191 2431 2191 2451 2741 2341 2401 2751 2721 2571 3011 2031 2071 1841 1581 2111 1361 2121 2201 1721 2291 1741 1851 1281 0611 1531 1201 1461 1161 0981 1701 1781 1791 1991 1331 1301 1291 0411 0131 0009931 0311 0641 0651 0531 0731 0351 0751 0609521 0441 0431 0081 0111 0651 0121 0781 0031 0171 0421 0219251 0019479981 0239971 015937903905913920903892929890968909900930829921890886919880882854911909950868876921980927911879854871889897888849900919851881863876893862925877796859790833862835762776812787793792771777796781785740765738789796721727699753765792801791810787828780840782786846762773763728716731738758691762713681720726700756723682759761737752750800741726741641749732833709710701681691704717673658658681672687637625663685654632675647683666665650641673643658654647670597671700646641651682673711613645635638664630706639664671640598603644616610569575585629561568518545570561600618618557564565568590560567554614513559525561562576523565501527518521519500510517512491541473465509513536592531502524486484497525478479448497504478507473471466483443466484433457460451467458435463487422466439448445430387423432413434440413408399380424412419431478404433375418387409390377391379374371374379369409403385411380416399375404441408413372399404380421396385345404407384397364372396393347414337384382381345357337349345347356365339354346331347311318352322304310313300327319337330264288301327309278320355320287318321330287276314291282304317315296286294330296261293287305319303276287270277287315285269280263291297288304308268260307281310285278268290273297301316308331279305311297291287305290287317291292293265309261271267292277307309258295305313277284279259295246299268266280279268276248279268244250265250266245231225239248250233241239244241234234245242239256230246248230231253210269229248251237257253234224247260222229216227252232207237233247223242229234217230221223214216225207209237217219213214204200217195222214211222206217198190207214220210213203220220240210234242208218198184246184206222194195214204173222188207219195198202186183185203206201204179179196206187184163163199181213188163211189185178248 262100200300400500600700800900>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.

1 786 871000000026 642 814000894 394 322000000000492 615 3870000552 989 13000001 161 906 45500002 442 444 43000011 826 771 80700510152025303540Phred 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).

99.4 %114 543 74299.4 %0.6 %

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.

99.2 %114 260 54099.2 %0.8 %

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 %283 2020.2 %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 %57 614 40850 %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.

97.4 %112 228 06497.4 %2.6 %

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.

5.1 %5 883 1205.1 %94.9 %

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.

5 741 058129 21479 976147 903113 433117 687135 163174 74279 051129 67563 36754 50478 26084 75646 39298 92974 17679 749105 242138 433147 473138 432162 414121 484196 147322 27623 754564 62734 88433 20868 19167 85531 27079 53533 62632 97455 45971 22419 496105 7831 566 61370 38369 950112 71894 626175 437149 868231 300357 46244 93458 19751 35665 94835 21156 01060 57045 095155 38848 24989 969102 461 041051015202530354045505560Phred 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.

99.75%99.74%99.74%99.73%99.74%99.74%99.74%99.74%99.74%99.74%99.74%99.74%99.73%99.75%99.74%99.78%99.76%99.73%99.79%99.74%99.74%99.74%99.83%99.74%0.25%0.26%0.26%0.27%0.26%0.26%0.26%0.26%0.26%0.26%0.26%0.26%0.27%0.25%0.26%0.22%0.24%0.27%0.21%0.26%0.26%0.26%0.17%0.26%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped