European Genome-Phenome Archive

File Quality

File InformationEGAF00000904166

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.

43 170 25530 682 34715 881 3008 690 5714 021 1132 614 3501 392 7211 085 942680 614551 562395 931329 706259 617220 476183 880156 032134 220116 027102 55489 40278 67970 48564 16158 61952 53548 22144 50541 90637 95835 67933 88631 88129 11528 07926 60525 46224 11422 75321 31320 16219 45418 86417 68817 26116 96716 24015 63815 09614 71714 40413 95813 18612 90712 25612 48312 08311 88811 27111 12610 81310 70810 16310 10210 1609 7519 5259 5889 4779 2168 9129 0568 6948 4428 1848 3028 0238 4498 0458 0307 8567 6137 5327 3367 2977 3377 1377 2607 0087 0546 8226 6786 5976 5856 6736 6426 4186 2206 4306 2626 1135 8726 0636 0206 0926 0035 7855 8355 6985 6075 2705 4375 3655 3785 3795 3035 1755 2115 0235 0094 9885 0245 0515 1044 9964 8864 8764 7014 5994 6004 5104 4244 5754 6364 4274 4484 5204 4114 4264 2844 2914 2974 2874 1634 0774 2084 2144 2034 1674 0614 2013 9464 0194 0563 9343 7103 8873 9193 7793 8243 7653 8283 7733 6993 6953 6593 6313 7703 7913 6653 6173 6143 5993 6433 6013 5273 4943 5433 5233 4423 4433 4913 4893 5043 3833 4453 3383 3983 3313 3923 3343 2763 2903 3393 2433 2233 1813 0623 1753 2083 1183 0663 1603 0363 0983 1313 0592 9622 9142 9432 9262 9152 8332 8862 8652 7852 7902 7642 7432 7842 6112 7272 7332 7892 7062 6582 7672 7292 6942 6532 6892 6972 7402 5862 5962 6182 5342 5142 5832 5402 5172 5142 4822 4672 5052 4142 4332 4582 3702 4262 3342 4762 4012 3642 4842 4292 3642 4312 4102 5072 3662 3542 3892 3312 3082 2552 3222 3562 2692 2542 3402 3172 2832 2522 2172 2842 2302 1932 2002 1502 1002 1692 1272 0662 0722 1312 1732 0342 1042 1022 0792 0782 0061 9701 9481 9231 8891 9371 9391 8861 9501 8541 8471 9721 8401 8631 7991 8141 8741 7581 8301 7451 8421 7661 7131 7061 6881 6931 6291 6831 6671 7081 7021 6061 6831 6871 6471 5951 7091 6751 6391 5321 5911 6591 5821 5931 6371 5451 6111 6541 5261 6111 5431 5271 5931 5101 4341 5601 5071 4951 5161 4511 4621 4661 4671 4511 4901 3821 3781 5081 4581 4321 3771 4771 3991 3851 3981 3791 3671 3531 3431 2831 3811 2971 3341 3471 2511 3661 3671 3091 2621 2741 2541 2411 2551 2611 3131 2811 2861 2791 3101 2281 2481 1871 1951 2241 2541 2381 2301 2761 2211 1621 2111 1451 1641 1691 2161 2031 1721 2261 2171 1441 1661 1271 1311 0801 1391 1211 0841 0601 0521 1131 0181 0541 0211 1071 0441 0769981 0641 0271 0431 0519929479809659329689461 0279649741 0011 02193493187787790291686392888091288790184890786485186881383482085791182783180582880281481082783980081875882780277879382577577376982077675779175775370672169971976076277273774277076169174764971772475270567776069665970567971067468668363068367464070463766769264468063261564760961764063963361060863961762063557061265458963959761156362360657353960960550958056854454649151853751750547752052252453952553053550948550650251953350149150750448050551149744747244148546148748646450651447544548245149143645244542345244841145441942240242541943740639944841942243242040340741642441442442841541539542038441441038438740236437539437738737240540637037938236638936538634434537636635835235733637135232837232932433734234033134034330335237636134830929832429036429729729130630032429730025226226024026226026728225330727026425426826027829323727925826927328330126728925025127127823927624024628825723124124523625722925027022925323323825722321624823721521623322519821721423922621120722418421322021719122320317219220718917121121219320118217618618718820018818920618018118819018421221320719022417320219620919920321018416718215216318718315716015119117818217317017118317320518716717417614213517116115015717315915118815614215313914916116214214514815614816613915515215015215816616716516313915013215313313812912013112812313414913214313512213713114312515011314111114212515813814414914412913413513314314913014215312415012512716411212713711413511513011111612112412811210111810212311312311910313312112311812213110512713811811214011710912512910310612310810912210911798120991131221171011229891114120109114105831091008979909796871121101221061011189611295106115106100101104958397868186888777789022 299100200300400500600700800900>1000Coverage value1001k10k100k1M10M# 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.

104 6480000640 036444 3522 345 0572 001 384764 3741 070 606422 562402 766961 514342 808925 985804 588877 2651 565 659930 2121 148 252834 4751 658 8552 169 2552 541 2682 819 9534 447 8314 461 1814 442 7694 405 09010 339 84116 477 61610 032 97416 500 29434 782 45740 597 65025 803 66669 836 28751 208 41885 831 680100 642 941206 365 48100510152025303540Phred quality score0M20M40M60M80M100M120M140M160M180M200M# 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).

97 %9 209 56597 %3 %

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.

96.3 %9 146 05096.3 %3.7 %

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 %63 5150.7 %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 %4 746 34750 %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.

94.3 %8 954 12894.3 %5.7 %

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.

44.1 %4 190 34944.1 %55.9 %

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.

629 2869604942 0269231 1038401 4111 77710 21410 2753 21517 8473 0322 03348 6124 51313 03516 4394 67528 10369922 79785 7526418 192464504496586 4247209101 0021 4061 1661 57643 113320 1275 5361 5286 9984 9761 11410 8861 9822 73038 3264 6645 0866 53010 5304 94413 96610 86816 60028 64260 8767 379 110051015202530354045505560Phred quality score1M2M3M4M5M6M7M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped