European Genome-Phenome Archive

File Quality

File InformationEGAF00002849279

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.

1 674 9142 092 0013 180 3245 201 5888 257 38112 366 81317 438 00123 415 85530 254 59438 444 84348 514 17360 975 43476 300 84494 395 203114 481 935135 163 486154 898 671171 861 762184 474 429191 657 129192 726 189187 890 313177 766 531163 455 590146 300 064127 574 531108 480 44090 135 01673 280 37458 294 91745 457 48734 824 58926 224 06719 463 93314 242 66610 339 7867 445 3285 352 4613 864 8162 814 5962 093 8011 590 3951 252 2531 012 097850 633734 844659 046596 328549 851510 988480 751455 007432 253410 037391 011370 960355 715337 729323 320310 873296 471283 171271 375259 273248 326236 624227 265218 684210 523202 507193 402185 737178 707171 043164 528155 335149 573143 465137 343132 050126 312121 593116 518112 825107 445103 48799 53295 84992 99489 00486 78184 41383 06080 83379 31076 95675 38774 01772 43071 06369 82468 68168 12367 28366 10065 35063 88063 63762 83361 92861 04260 21259 86057 99457 45455 83655 62354 53652 81751 23550 51649 70748 99047 89947 29145 67845 14543 66442 52541 56239 91538 79037 94037 29835 66534 88134 23732 78931 93330 90130 27929 21028 37527 54626 84025 69125 26324 85224 06323 62123 06022 31321 81021 57420 73119 85919 75619 26918 75918 65518 28017 84717 47117 33317 11216 45816 08815 68815 72515 35115 34015 11514 94615 05014 32414 12413 91113 44113 44413 17813 24112 76912 91112 81012 29612 34412 11812 09011 84211 92811 59811 18411 23711 10811 00210 81610 70810 55110 44210 21510 05910 0549 8269 5719 7919 9149 9039 6149 5799 3479 1939 1319 1529 1899 0128 8558 7568 6058 4308 4418 2698 2718 2688 0968 0507 7107 7647 6697 4957 5507 4047 3767 4887 2327 0137 0327 0816 9627 0476 9876 9296 8506 9036 6276 7786 4396 4736 3856 3906 2676 3606 3946 2755 9986 0506 0045 8935 6865 9495 8695 6965 7315 7765 7155 6745 5725 5735 4945 3705 4255 3715 3975 4235 3725 2935 1375 1545 2535 1945 2915 0895 0505 0505 0284 9805 1535 0284 8844 8724 8744 8144 7824 7504 5554 5414 7414 5124 5854 5514 6534 4254 5524 4554 2804 2974 1334 1814 3054 2344 2854 1214 2104 2044 0813 9603 9063 8273 8103 8833 9223 7383 7893 7443 7593 7983 6323 8023 6983 6073 6003 5053 4983 4083 3753 5933 3983 4763 3423 4113 5023 4063 2393 3303 2803 3323 3533 1523 3873 2493 2953 1983 2943 2313 2213 2133 2053 0843 1933 0763 2283 1813 1423 1203 1463 0723 0503 0523 0423 1673 0252 9522 9183 0352 9852 8782 7843 0352 9432 7632 6832 7252 6392 7022 6482 6842 5852 5852 6472 5692 6252 8132 6752 5772 5912 5392 5542 5542 6232 4902 4572 4342 4392 5332 4892 4672 5032 5012 3692 4852 4102 4082 3792 3862 4252 4152 3812 3872 3332 3662 2692 3722 2892 3792 1732 3672 2482 2322 2722 3292 1562 2522 3162 1562 0942 1132 2202 1062 1322 0962 1342 1342 0892 0862 0202 0882 0552 0352 0732 1142 1622 1472 1572 0962 1142 0782 0992 1012 0352 0641 9601 9352 0331 9821 9561 7841 9632 0621 9661 8371 8861 8781 8421 7741 8751 7571 7921 7771 8621 9231 8531 8421 8561 7671 8271 8211 7681 7881 7261 7701 6791 7071 7281 6301 7131 6561 6241 6571 7341 6791 7101 7511 7151 7201 6361 6421 6971 6941 6641 5741 5611 6261 6081 6391 6151 6641 5971 5921 5901 5711 4841 5131 5201 5051 4921 4931 5331 5301 5071 5271 5241 4741 4981 5471 4641 4811 5671 4001 5811 4821 6101 6661 5641 5401 6741 6961 5211 5801 5981 6161 5711 5911 5061 4991 5251 5271 4941 5591 5461 5421 5981 6071 4881 6031 5971 5051 5591 5581 5421 6171 5631 6191 5611 5021 4311 4311 3831 5501 5371 5451 5011 5261 6371 5081 5671 5861 5561 5251 5151 4831 4611 5801 5301 4521 5151 4451 4791 4131 4431 5121 4161 4601 4511 5061 4611 4061 3891 4201 3221 3751 4011 3271 3001 3711 3371 3011 3491 3021 2891 2301 2391 3001 2931 2191 2101 2231 2331 1481 2341 1511 2581 1691 2241 2701 2091 2121 1521 1311 1641 1211 1641 1341 1571 1521 2001 2131 1431 1041 0861 0741 0651 1221 0401 0651 0161 0831 0881 1201 0401 1151 1589971 0511 0921 0351 1239771 0751 0269791 0451 0121 0429939729969989319988919859419609951 0111 0171 000939987970955891973966999913957954954935899868924908899957927941871968912877953878915929848912928819818839952892909894887844900854890880895889835854841791885828825849796855848818780834836833808823798831824831814813834808786767787784793767825799825805866826785784878865783836789789775787749791793789808768811780704783761760721812735726766755764728715732741699741718684744673709682693703703660726690698673673716676669664678674657680659658641649640673697693681669665658671680669664665693691712703680705654706630669707655661656676624636648631637664646671698689691660653634616607640645710645659604625585606643614641610599614627594585611584550560603607601609655599601608621613563569598609610585611583538562600576587589549603559589585565588614545561593599596584553594591593575631596536570582598571611546564614618582592576579585539582555551598565564587556602561561565623554561601541521588581587534592528556551544512549528521516628555598545607544571601696 732100200300400500600700800900>1000Coverage value1k2k10k20k100k200k1M2M10M20M100M# 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.

3 597 9510000000122 334 9780002 857 112 1070000000001 631 441 91100001 803 463 24300003 977 043 24000008 795 961 15700046 276 299 78900510152025303540Phred quality score0G5G10G15G20G25G30G35G40G45G# 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).

99.8 %432 554 23899.8 %0.2 %

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.

99.6 %431 778 24299.6 %0.4 %

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%).

0.2 %775 9960.2 %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 %216 778 98850 %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.

98 %424 752 66898 %2 %

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.

11.6 %50 173 03911.6 %88.4 %

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.

20 255 051501 180310 238580 344455 942470 634510 400720 956302 740522 886251 714211 916295 700341 263184 299393 456287 710317 407427 052610 089668 912574 037668 189490 196776 6831 286 84186 5862 238 201135 085127 578252 385262 543112 311308 018130 784126 328207 293290 24472 511421 3986 134 259262 419247 692413 049350 501657 509546 901907 7231 289 804160 426206 039192 997231 971116 541196 793216 172152 708579 754161 267325 648384 309 781051015202530354045505560Phred quality score50M100M150M200M250M300M350M# 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.

99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.81%99.83%99.82%99.81%99.84%99.81%99.82%99.81%99.88%99.79%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.19%0.17%0.18%0.19%0.16%0.19%0.18%0.19%0.12%0.21%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped