European Genome-Phenome Archive

File Quality

File InformationEGAF00002797838

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.

88 121 54193 123 76733 436 18932 526 70516 716 23314 394 5439 209 2947 681 7335 629 1254 708 0753 759 2643 229 6222 699 6142 390 2552 066 8911 859 4941 652 0371 507 0051 375 4611 275 9351 163 3681 095 5761 020 649970 278902 012859 635816 306781 696741 985707 124677 594651 783627 459608 365583 948561 267543 345529 936510 437492 181476 248462 650445 274434 471419 368411 439399 589389 121376 651368 758357 609349 700343 245335 552327 227320 617315 176310 640301 117293 717286 264281 997274 572269 814260 599258 747252 628248 658246 110240 919235 310230 590226 588224 514218 724216 508209 414206 506202 289200 675194 061194 903187 887186 802181 517177 725174 692174 248170 355167 204166 836162 966158 687156 223153 359152 630150 852147 758146 096144 040141 403141 393138 568134 480133 030131 689129 048126 345125 386123 099122 462120 944118 253118 532113 662113 010110 621109 615106 424107 272105 045104 987105 225103 301100 38998 34197 91096 96595 72493 55791 29592 02489 84090 19087 83387 02084 95984 54182 34883 55880 90780 19978 56077 81576 57675 59174 67074 02072 39370 99870 14369 46868 66467 48767 11965 56165 22264 68264 90064 46062 57762 05861 64961 40660 82660 45060 21957 83057 85757 79856 93356 90355 79855 17853 60152 98852 97752 41551 33750 95849 89149 19648 64148 63447 69347 65647 41147 05345 99645 23146 28544 58243 46743 06043 45142 65442 11842 14541 10441 45740 75840 30140 09140 07538 68638 80338 19737 74837 59737 06037 40836 68636 13535 69536 22035 37834 62634 80033 81234 02032 82333 06632 92332 96731 99632 15731 88030 60930 83530 83430 68929 76130 02129 29928 72928 46127 90727 57527 76128 02027 87627 06727 39326 47026 44225 91026 37526 06125 48025 12525 37825 34424 61924 26524 39824 22123 71323 21123 68423 07023 09922 55322 77322 31721 84921 87721 77822 25621 12821 16820 67020 52520 57720 89020 53419 82619 89519 44619 92319 46519 48419 24719 40418 89918 76719 12418 61917 99818 28018 23417 73218 17417 77416 92017 80416 90016 95617 20716 98716 63916 75516 57316 35215 92616 05016 02316 25716 27616 11615 60315 52515 38314 76515 89715 64115 02814 54914 19514 53814 45314 24414 70214 27213 84714 42113 92414 11813 67613 16313 55313 71613 65213 12713 36413 09613 17013 13012 98012 43412 48312 35512 58212 42112 71312 37412 04912 10112 40112 20112 33412 05112 21812 06711 84711 60611 59411 74811 71611 31211 38511 43911 14111 29211 20310 73910 82610 75710 79510 62810 45310 60310 06810 07310 00010 1119 9959 93510 10210 0769 8249 9279 9489 9749 7189 4559 4019 3269 6419 2249 5119 4029 6169 5749 2839 0889 1868 6308 7548 8078 7148 4948 5088 6348 5848 6478 6798 4688 4308 1278 2798 4608 1998 2278 3738 2307 9648 2337 7537 6047 8957 6077 9127 5817 6407 6397 4107 5177 3647 4557 4777 4857 2707 1917 3317 4247 3747 0706 9877 0116 9287 0356 9986 6996 7976 6296 5886 7096 6896 8176 5866 6246 8376 3936 7666 6176 3426 3346 4046 4106 3786 3005 9336 1116 2495 9315 9135 9145 8935 7585 8235 9215 8005 6445 6985 6585 9055 8405 7265 6445 9075 6475 9125 9195 5875 6695 5095 7435 3665 4695 3785 2915 3275 3725 3445 2215 4075 1555 1184 9844 9094 9364 8794 9274 6314 6964 9184 8475 0014 8994 9044 9425 2974 8474 6544 9654 8024 6214 8844 6144 7464 6154 5494 4274 3874 7364 5194 6394 5834 3574 3154 5004 2494 3904 0004 1214 1983 9794 0524 2124 1094 1154 2084 1804 2624 2324 2344 1364 2434 2264 1994 0403 9523 9843 9573 9083 9733 9524 0243 9323 9993 7983 7943 6073 7213 9194 0683 9103 8564 0013 8673 9413 8433 9133 8943 7493 7913 7673 7893 7733 8043 7103 7383 6743 7533 6823 4673 5093 6713 4123 2593 2253 4103 5153 2863 3053 4333 3703 2313 3423 3203 0873 3043 3503 2793 1363 1523 3363 0883 2153 2383 2223 1243 3123 2682 9443 0043 1692 9722 8992 7802 9152 7872 8682 8132 8172 7882 7642 7472 8263 0172 9292 8142 7012 9982 9302 9053 0152 6423 0532 9062 6812 6082 5652 6042 5882 6692 5772 9542 7492 5412 5932 5752 4362 6542 5952 7262 5772 5342 8722 5772 6552 5322 5352 4282 6052 5282 6192 5162 4992 4172 3692 3772 3032 4292 3012 3872 3762 3572 4132 5432 2892 2992 2662 1892 3452 2162 4002 4362 2562 0732 2152 3322 1722 1742 1862 3232 2712 2072 2492 1212 1782 1172 1012 1812 0322 0531 9842 2852 1192 0462 2882 1342 0692 1002 0512 1062 2302 1341 9301 9841 9902 0882 1842 0062 0231 9702 0161 8912 0961 9551 7941 9391 9341 8882 0781 9651 9391 8831 8291 7791 8751 8341 9781 9201 8961 8521 9111 8041 7921 8691 8671 8671 8131 9401 7911 7851 9011 8191 7061 8961 8251 7661 8911 7531 8201 7531 8281 8291 8181 6891 7421 8021 7581 6441 7161 6381 6311 7381 7451 7101 7101 7611 7441 6651 7411 7071 8111 5331 6581 7831 5701 6331 5801 5271 5931 6221 4931 7571 5931 4481 4471 4541 4911 5071 5451 5211 5451 4471 5681 6961 5761 4221 5231 4681 7551 6011 5151 4251 4841 5261 4591 4001 4291 4281 4451 4181 3591 3911 4281 3671 2931 3721 3481 4241 2691 3611 3271 4421 5181 4251 4571 4441 4471 3611 4501 3831 5661 3271 4391 5091 3401 4411 3041 2861 3201 4041 2451 3241 2711 3031 3031 3201 2391 3461 4121 3841 2391 1871 2381 3981 3391 3271 3191 3191 2181 2461 3821 2121 1891 3621 2791 2061 2271 2721 2701 1941 1531 2171 2041 2531 0961 1971 1101 3711 2911 2951 2161 2721 1411 1691 1541 1571 1781 1661 1001 1871 1891 3171 2161 1911 1011 1201 2931 1541 0731 0611 1441 1751 1581 1471 1601 2121 1301 1751 1331 1251 0861 0431 0591 1021 0801 1341 0751 1411 0771 1131 0581 0691 0491 0521 0921 0581 1991 0821 0271 2379711 0151 0381 0711 1141 2281 0441 0141 1741 0349911 0101 0339751 0411 0641 0769559631 0729491 2271 0671 1271 0229399279869559599091 0119319849871 0539509359561 0291 0231 0601 0109009099049169839928991 029888922953678 594100200300400500600700800900>1000Coverage value1k2k10k20k100k200k1M2M10M20M# 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.

0069 19300000000164 068 9390000000000000345 478 451000000000009 146 445 95100000510152025303540Phred 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).

100 %63 947 434100 %0 %

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.

100 %63 947 434100 %0 %

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 %00 %100 %

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 %31 973 71750 %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.

100 %63 947 434100 %0 %

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.

0 %00 %100 %

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.

246 938960 2042 645 70862 288 584020406080100120140160180200220240Phred quality score5M10M15M20M25M30M35M40M45M50M55M60M# 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