European Genome-Phenome Archive

File Quality

File InformationEGAF00001159916

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.

269 055 00673 005 72718 802 8549 216 8035 439 2534 402 0233 702 2093 296 4312 994 1962 768 7272 584 3882 432 3012 294 9722 182 2472 074 4751 981 4491 901 3261 831 9531 769 8431 705 8361 646 5931 589 4251 533 2971 482 5281 436 7481 391 1881 347 0201 306 9751 262 8951 227 6471 186 5631 150 8231 114 7651 080 7571 046 8801 014 370981 916952 563920 079891 760859 724833 217806 089780 266751 092726 848701 034675 174652 305629 624607 131585 675566 410545 839527 021509 326491 630471 770453 947436 225423 178405 382390 136376 423363 810349 942338 334326 400314 616303 395293 020282 062272 860263 519255 376246 360238 387229 761221 442213 120206 067198 987192 552185 503179 091173 162167 637162 882156 880152 678147 582142 623138 964134 257130 825126 099123 265118 098114 883111 962108 874106 030103 00499 64496 86693 61091 02587 82985 69583 32580 86878 15676 02173 37372 26369 86367 85165 91364 20762 00460 95258 85857 44055 43154 00952 81251 07149 97548 09046 55445 66144 67043 40842 38341 05240 04638 71737 68037 34335 92535 59934 61133 65132 83432 07231 09530 74829 83928 95227 97027 31626 81026 00325 43525 06924 18123 52223 28722 75022 07421 61321 02720 66719 98919 47019 04418 64118 19117 91917 62917 19816 74616 37316 00815 73215 45714 73214 44514 14614 02613 41613 50613 28213 09112 40912 38211 97411 65911 26711 14111 15410 79510 71010 48410 15510 0129 6269 6328 9248 9148 8488 5628 5268 3218 1357 9387 6697 8077 4317 5037 3427 0677 0146 8426 8496 7756 7306 4396 4196 3226 0915 9705 9425 8105 6495 6295 4575 2745 1284 9184 9554 9784 9264 7664 6554 5894 4494 3134 1834 0834 1434 0514 0043 9603 9243 8903 8613 7753 7563 6613 5593 4463 4703 5033 3893 3573 4313 3273 2613 3773 2173 4053 2563 1483 0282 9592 9032 9402 8202 7622 8342 7852 7332 7402 5232 5312 5442 5602 4002 3422 3712 3362 2812 2532 2382 1882 2622 2132 2412 1382 2612 2002 1232 0542 1502 0801 9772 0282 0081 9211 9211 9371 8451 8251 8511 8571 7801 8341 6951 7941 7641 6861 7071 6951 7181 7071 6561 6821 6691 5831 5761 5681 5391 4521 4951 4931 4911 4421 3741 4051 4671 3791 3901 3161 3361 3051 4011 3471 2721 1851 2531 2691 2051 1201 1921 1621 1691 1221 0821 1221 0541 0151 0381 1049751 0251 00998897998398993090492685192282883583084383785588282379979774380175677177275771071970773371771567174365972974467766668774771472670267169470069469567669166664265166158763766968564462864963559262665462762261558861857058854559158956156157351652255750850054353453353848948750252448145947745845546047947242745642244941740642242245143141639141441639939539240144541139033038639239537036938035534830135135730534335732231933633132528931131934728934434131931930433632633134332834231929831331332930233130432131031631228631131231232728526331431129029029828727429025925729629125626326327326128525925025926227624923329027026523523922924626026621926219922222924320525019123420821922521421921719922818120321219021519018719019019218321217820318919721218219619619419421620218316018818618716418116716718418514818017316418216317419817520115818118218714818617015916115015616514516116915316216117217915916116417416716817415212816916216017516517316415416816414915716817915914116614715717214917015219016517016517516014316415317115317716515315714016515017516016717215816717916916819415716119916216918416818118916318218119616717719919918118515916518815815617018416713916514315119215215816415916015814915416716014615815514815013514516313511914411712712914713214214112511511412395137136132118117123135118122128114116142123104114132105124110106113120113981251061111179510410410110210410510785939683899010490106889384909690778691869586791018087878884837574856979698458638081696774605958537067625655537251664450644963495755385449374146475434506544556150414049466245464337564137565755493940455042483639374245394245404735364648283731372526332721282921292727202733292930192835343430263438312927323529362924331919233435232825224014352337342936321929332729263128362020232718162611 971100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

734 60300002 909 1611 895 6219 413 5757 858 0892 749 1484 023 7291 721 8591 793 3823 861 5571 420 8903 864 0533 383 5273 766 2196 257 9243 553 3325 143 5313 543 8556 900 0039 270 09210 263 20211 873 49219 094 52918 768 03118 547 02218 790 40743 724 94571 625 21443 329 52275 345 959179 873 986225 772 343122 751 446366 607 554225 966 458447 735 777491 420 3871 292 214 57600510152025303540Phred quality score0G0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G1.2G# 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).

99.3 %49 905 59299.3 %0.7 %

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.

99.1 %49 761 39499.1 %0.9 %

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%).

0.3 %144 1980.3 %99.7 %

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 %25 118 46050 %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.

98.9 %49 702 52898.9 %1.1 %

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.

13.8 %6 944 78513.8 %86.2 %

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.

5 399 9095 5894 0849 6474 97911 54110 88522 96318 77157 25741 41214 20791 09718 32516 170203 91036 98582 10775 82429 399155 4222 16198 872343 7711 96914 2142 1812 1882 373952 6085 4354 6985 4967 2666 9749 752216 761651 12717 9424 94831 91617 6562 58048 8123 4185 180133 7185 9968 1488 47017 6649 58633 03429 87847 06084 136197 58040 890 869051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M# 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