European Genome-Phenome Archive

File Quality

File InformationEGAF00008412693

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.

68 774 531129 474 212196 980 509257 607 972299 640 626317 377 504310 429 157283 733 739245 020 054200 834 894157 642 452118 917 49986 707 17461 329 67142 237 74728 409 88518 781 21912 227 3797 939 0985 145 6673 366 5932 239 9341 549 7771 108 022839 832656 827550 092472 177416 431374 852339 409314 382291 857269 239251 530236 222219 111205 850194 627179 974170 184159 932149 924140 956133 908126 064119 467110 516103 60097 26891 53986 69280 51075 87771 45166 62063 05158 76156 45553 54850 07948 35645 21143 40041 24838 97937 73035 77933 98133 01331 65030 86829 18928 37327 90827 00026 45225 21325 08924 14222 97622 38722 15621 59920 92620 69920 58919 69318 93118 90218 28617 75016 97816 82916 74816 38115 80815 55115 30415 19314 66414 31913 90113 76813 43513 27212 89612 79312 64412 44011 97611 77211 68511 60911 18111 11110 69210 55010 1009 9629 9389 6319 8749 4709 5219 5599 4109 2309 0748 6068 2908 1598 0077 8757 9067 8417 8067 6207 5957 3217 5687 1727 2076 8866 8447 0416 8226 8196 7106 5806 3556 1666 1826 0165 9295 8555 8815 9055 9205 6565 7295 6525 4725 4605 3475 1715 3015 3265 0085 0664 9405 0244 7314 5524 6634 5684 5214 8034 5704 4414 4274 3204 4334 1044 2724 3794 3044 1954 3064 2284 2904 1683 9964 0654 1324 0574 0273 8643 9983 8503 8063 9153 9253 8373 8393 8013 5573 4883 6003 5373 7303 5173 4813 3753 3243 2383 5033 3683 2683 2823 2623 2353 2823 0793 2273 2133 0663 1063 0972 9683 0572 9063 0062 8782 7202 7372 7222 6932 7142 6992 5952 5642 6162 6152 4312 6092 5402 5622 5292 4812 3482 2302 3122 3062 2622 4452 3302 3192 2312 2412 2692 2252 1862 1242 1022 2422 1272 1462 1062 0472 2042 0722 0302 1242 1192 1502 1242 1332 0631 9752 0992 1632 0412 0221 9631 8601 8281 8571 8381 7261 8331 8291 8451 8821 8641 9121 7121 7961 7531 8671 8111 7301 8161 8231 8391 7581 7181 7301 7041 7821 7471 7271 6301 6861 7021 5881 6631 5861 4961 6491 5821 5791 5911 6181 4871 5121 4791 4871 4631 4541 4891 4871 4901 5381 5361 4811 5771 4841 5091 4331 4521 4351 4721 4521 3611 3231 4371 2991 2971 3511 2701 2221 2081 2431 2351 2001 2541 2501 2281 2021 2561 2561 2091 1941 2481 1901 1501 1441 0981 1771 1961 1731 2021 1231 1581 1791 1321 1241 1251 0981 1851 2731 1531 1961 1501 2281 0291 0641 0971 0451 0641 0601 0381 1281 0961 0731 0691 0741 0321 0701 0571 0411 0751 0881 0689591 0311 0711 0339881 0161 0251 0221 0261 0069991 0221 0331 0141 0199961 0031 0581 0409461 010943941954942940900860837888884852907897887868840903932914837880871918858920871862918946930853877908862897879931875915888828849769825836787872879828839757785819873763861843774789767834729777721751762786741634729693735676725768796790738708717679699651655663679665669710697653646657689688716717661680640651770706674687692627651636601625661580642635606633609578541618578600626643583603628681596586603573601611613589559660574580579572593580551524574513499571551515565518514564593505576569570528553528526531462538542525536511487533491558511501501524551543513543532511514534516494552538525507544484511482506465513523535555553521534507543573550494509535510490532486489446501491524568473500454509498485547504492505439457486447471478476426402482437477472433439418434453477426412433420414423407417448404392384383373409420399421382417422438433467480431424433453429407431420481432425412405424413421371410395412400409401392389367404403394393363400406381378386383423403371368387351358331377406361356324323332363362346315362388351340405389357363389321321386330354333350365313319367341309386357352323360325335329323364337370354335288315304329320325358369320320348334339313356373325376324353337346339353343331305337301356323301362334298329365318339312308309329306292313339346291348303296297293315327307300306260267296299325293283272324305291306339274331309332343349284301310333322318312294327306322312312292320330354317314340334337302332323316325297347347285321329318364285315309335309268305282299271287316300308291303302275296320317347337312318318303291356332325310294289294301299269301267307291307257264262300284276273310298269265289263260300262289268270269259257292262292297287255274275273281276277295288274263238284264263280259265224234316 313100200300400500600700800900>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.

2 435 597000000054 321 760000787 430 452000000000464 994 2920000562 317 55200001 279 368 37800002 716 696 72200017 101 606 66500510152025303540Phred quality score0G2G4G6G8G10G12G14G16G# 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).

99.2 %150 836 66099.2 %0.8 %

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 %150 624 01299 %1 %

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 %212 6480.1 %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 %76 056 85950 %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.

97.4 %148 201 14697.4 %2.6 %

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.

20.8 %31 644 12920.8 %79.2 %

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.

7 918 279149 86990 987184 241135 549145 596153 284224 54096 286167 95373 18063 69986 406105 24051 149126 53989 521100 766126 026185 444192 807186 183218 968167 887273 740472 80626 044815 15339 92138 15178 38987 19239 019103 84441 52341 25766 46295 06322 603141 5932 016 22891 47182 323148 035119 836231 386197 704310 225511 85251 51872 04661 63881 23834 10764 33470 24648 628207 11448 094108 602135 219 137051015202530354045505560Phred 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.

99.86%99.83%99.86%99.86%99.86%99.86%99.86%99.86%99.85%99.85%99.86%99.86%99.86%99.86%99.86%99.86%99.86%99.86%99.87%99.85%99.86%99.86%99.89%99.86%0.14%0.17%0.14%0.14%0.14%0.14%0.14%0.14%0.15%0.15%0.14%0.14%0.14%0.14%0.14%0.14%0.14%0.14%0.13%0.15%0.14%0.14%0.11%0.14%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped