European Genome-Phenome Archive

File Quality

File InformationEGAF00004857150

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.

46 152 997100 048 511172 934 459252 114 270319 038 772357 027 422358 250 461327 540 089275 332 490214 822 172157 126 574108 489 39171 210 88044 743 58927 205 78616 094 5509 426 2715 553 4213 353 4522 124 9761 443 6671 049 041813 443663 505559 046486 153430 526384 241345 624313 011285 646258 967240 650222 892206 924192 212177 827167 371155 293144 895135 627125 616118 529111 528104 49297 80091 64587 93780 27476 87271 20166 44763 13259 16555 59052 95749 55246 32944 94342 56741 08638 77037 58536 31634 76033 38932 54631 75430 33529 75428 77127 99827 15327 05925 60325 52124 43523 93923 13222 69321 62121 17420 72720 11019 31918 52718 36617 80617 87017 11016 35916 23415 81615 59115 08414 43214 26714 20713 77413 42613 08812 73012 70112 51112 35212 29012 04311 39611 60411 31510 98010 94610 80810 40010 0579 8279 3399 7259 4869 2409 1028 9388 8828 4548 4048 3188 3408 3998 4838 3338 2558 2628 0627 7887 6817 3977 3617 1947 1196 8076 7666 5406 7586 7226 5066 4386 4016 2316 2646 2216 3476 1496 0975 8375 7335 6645 6135 6315 4945 5075 5475 6085 2785 2615 2835 2105 1385 0695 1624 8364 9854 8554 9174 7914 8934 9974 8604 9194 8304 8694 5544 6224 4914 4224 4974 4124 3124 1814 2374 1754 1164 2284 2904 1423 9614 0164 1474 1944 1573 8493 9063 8683 7873 7203 7213 7133 7323 6443 4483 5073 5303 4393 3973 4373 4103 3703 1203 1623 2073 1013 1363 0643 0473 0822 9403 0172 8602 8493 0653 1212 8702 8752 8233 0152 9332 7412 8142 6712 7612 7092 7492 6122 6292 6192 6712 5472 5532 6542 5412 6022 5932 5132 4872 4972 4842 4202 4282 3562 4362 2182 2902 4012 2972 3252 3332 2712 1592 1162 1412 0272 0902 0742 1102 0671 9551 9982 0252 0522 0531 9341 9351 9981 8731 9721 9862 0291 8331 8921 8311 9071 8431 9321 8281 8701 8591 8471 8001 7651 7991 8171 8061 6841 7421 7221 7441 6641 7451 6681 6291 5681 5711 6201 7181 6441 6491 5541 6301 6001 6361 6291 6081 6651 6381 6191 5201 5801 5021 5021 4851 4551 5311 4761 4901 4501 4381 4771 3871 4391 4691 4351 3971 4831 5091 5421 5191 4601 4681 4291 3931 3921 4171 3281 3701 3611 4471 4631 4151 5241 4541 3861 4061 4501 3711 3211 2421 2891 2901 2971 2931 2541 2381 2361 2291 2741 2171 2041 2091 1501 1441 1201 1501 1601 1891 1971 1411 1191 1181 1501 1721 2321 2281 1581 1611 2561 2031 1871 1881 1271 0591 1111 1301 1181 1851 1281 0801 1291 1361 0881 0851 0309971 0001 0269671 0249879919851 0261 0041 0809951 0179229769989911 0661 0309751 0161 016972964963944943944974975964978933952957982922954928991925946923891857912892886879845931930938934909881866855879870830839836795836846809803827827798788794795709821758796737798751734811808819711758763733755756733731714793716687799745743731802760720735755737725732705720741740691723715780709682722704661642678660700748756726692709657697649667672681665667703709679672673635657721669654685646648688686659670638660641670696622675629661647689613592574623656620619589630605585609629618589581617580587591641645560559580563584587567545629566539591503550518504549514568519531518531525502543548553548545510518511558516478464501472494499488502492460485470510522519492469520459495473442441435462453472507436480460461491480476482483493531439471467420433450466474465491463441435463460492459472458462459472485446450449445448423452442426432468455469468451509474459461431481438463480438426465458468439458459470452432464456466419452434460474437425419443449418425433423387413379382410380417386390389422412399443432426423461490429365430402419399402353385399402365409459416410393377374428405405395412398413444386442442379381394409426355338385349369365355383365331317358381368367324354371379376343373368368347357351342369382369340353337326333302357372387341365345346335340391329364348343365349368334373379322350352348397354345345400370390377348360344346363362383374365374376391374341356343366359339338347343389351364378380348409384340350347370351347343373368347364358379406396371361348319344343312319299343306331328330309356333315329373315335296338320331316319338299278334317311333298314321307282287342267307307313324303298312300306308316295310338348316329319322320311294295290287305291294310297272245315275290282269260267250301293 258100200300400500600700800900>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.

6 192 562000000022 528 052000892 833 927000000000525 200 0900000615 029 98800001 361 440 97600003 134 502 30400015 908 413 59500510152025303540Phred quality score0G2G4G6G8G10G12G14G# 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).

99.7 %148 281 79599.7 %0.3 %

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.

99.6 %148 152 79099.6 %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%).

0.1 %129 0050.1 %99.9 %

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 %74 391 19750 %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.

98.1 %145 915 36098.1 %1.9 %

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.

6 %8 907 7126 %94 %

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.

6 921 188138 00984 067166 415123 102130 858141 435198 59385 365148 55569 04160 20381 86398 72550 890117 24784 32797 254123 274177 864188 405171 454213 921165 229260 730437 53724 410744 52836 67536 14569 73076 70733 62692 88038 70637 85359 87083 32021 626123 9991 842 85084 81578 073138 812115 658221 727189 618304 629472 20451 48769 71964 58877 98534 94765 06070 861218 117197 05150 156107 484132 967 451051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.91%99.9%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.91%99.92%99.92%99.91%99.92%99.91%99.91%99.91%99.94%99.92%0.09%0.1%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.09%0.08%0.08%0.09%0.08%0.09%0.09%0.09%0.06%0.08%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped