European Genome-Phenome Archive

File Quality

File InformationEGAF00002307286

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.

188 006 498311 313 159399 829 529429 407 938401 500 977336 107 303256 751 338182 030 695121 554 99877 148 76346 983 11127 786 71816 078 8669 249 1645 324 6003 156 3301 942 7681 262 703876 844643 739503 664411 661344 337291 465252 034220 665196 816177 138157 400142 194128 150117 357108 22099 37691 48882 50775 95470 81365 46259 47755 35051 11346 59043 88341 28738 19535 92234 81634 05132 47830 16528 60726 81925 31123 98123 63622 76822 63422 02021 00519 76819 39918 39617 91317 53416 90916 14715 64915 26214 47314 25513 71513 41712 90512 52012 02311 92811 45011 18910 77410 84910 78510 3989 6529 6529 3658 9389 0478 7058 6728 6848 3498 3478 0307 9887 8287 4417 6397 5877 2036 9696 9066 8026 4896 4526 5936 3506 1726 1806 0415 7636 0085 9195 8395 6825 6285 5255 2375 2575 1505 0204 9154 9314 6414 6714 4884 4394 5294 2854 3234 2084 3124 1523 9093 9913 8983 8613 5543 5203 4113 3213 3003 3013 3343 3413 0153 0782 9853 0133 0283 0593 0763 0583 0433 2152 9562 8732 8132 7642 7562 6552 7932 6022 6172 6432 5532 5272 3882 4392 3832 3302 3292 3012 2562 2372 2012 0782 1862 1992 1622 1222 2432 2442 1572 1622 1012 0071 9811 9521 8261 9772 0261 9861 9571 9311 8121 7511 8331 8051 9251 8921 8831 8741 9111 8511 8471 7741 6321 7191 6931 7111 6861 6721 6721 6411 7201 8041 6201 7231 6381 6451 7291 6121 6301 6471 5381 5531 5481 4961 4651 3911 4931 4061 4321 3851 4311 5691 4361 4111 3821 4411 3961 3721 4101 2741 3261 3151 3221 2021 3521 2771 4241 3041 3171 3301 2911 2051 2851 2141 2501 1731 2161 1381 1301 3471 2161 1241 0921 1471 0631 1281 0921 1851 0561 0701 0229831 0121 0341 0971 0879561 0251 0311 0071 0381 0049979771 0581 0241 0431 0231 0499688979179539249079499639119659581 0461 0479559439319171 005974883846923926915924904911909918841848896909884885817864894845853836846816871854788827866793803802782833808783771803789811842796755736803780781714696681685691702717654686653642748679679700664675710678693688736687676714718742734703735734693731718656674666698610649687691717712722671726664750643757809707754692702749729726756725752765760813718749729704745714728751704767759740792745746690748764717727685685671719703692751648700653655744677713755754732727736762706746731732746718693742692686649677708696742741698702722733712680616636645597641563555591594665640626614672627612610657628613580582572530555585528525528517548503534522512521540513485517517451432493421485428418433409429413433456468456458438382400389403429401391410377389373348325380373349411387359343376338381356364345321344360343334323305358317288298292274304337310321305309311301279288292290284286300313250274263248259330278286271257272245264295281258283265251278277300254288312293278306256288283238256229239279272274239260248275264240251268251236261236251252245251232234248218220252245231238247234237224233266241270261237254262260258239255247275277252239270252286246227242216235247269228191229232217219224224200229242223224242199222238219234221225206198213212222204228227246190219233194233233204227217224213203213201207216189260212202206212214260238225251220232201226212222220236228246212222253220240213229210243206219240213214179207197237207214205162209214208193177237197218214246229238250231252212245259220236259241221212199223237240227269264244244255224249242271255286250263261230238265254283303293269246271265256273279268280237263250234223266229259275295275259260236232219228247246249242225236243218220225218207228211223186194196235226246246260234227264258260218243251260243278254258272243272245259256225261228249255250251245265252241211224260239242226231239221221241207226208214226234252256226227214268242233220218217227213234219211225208209211222199231190214184184224178231214224190217201181203214207233219242218211224189223234260239246216209218243236252244240275265240202230223213210192209182161125 409100200300400500600700800900>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.

2 068 547000000089 601 736000740 408 614000000000446 616 1780000538 897 79600001 026 426 09800002 008 212 7880009 912 504 15100510152025303540Phred 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).

99.5 %97 308 54999.5 %0.5 %

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.

99.3 %97 095 30899.3 %0.7 %

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 %213 2410.2 %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 %48 889 85450 %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.

96.3 %94 121 19496.3 %3.7 %

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.

8.9 %8 705 1528.9 %91.1 %

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.

4 275 71573 92549 35991 70370 15275 11282 328110 49462 93184 44441 62233 80345 00048 39725 71760 21341 17346 45255 25872 90471 83284 698104 27680 357128 858219 22318 345421 96923 53221 23542 22842 64326 15952 60522 86223 27534 92544 63715 33471 731970 74952 40651 48087 24070 424130 540118 002175 888302 64736 86747 53741 90054 24527 54059 74748 67237 045123 32238 79974 29589 949 812051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M# 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.

99.78%99.76%99.78%99.78%99.78%99.78%99.78%99.78%99.79%99.78%99.78%99.78%99.77%99.78%99.78%99.79%99.8%99.77%99.82%99.79%99.79%99.77%99.84%99.72%0.22%0.24%0.22%0.22%0.22%0.22%0.22%0.22%0.21%0.22%0.22%0.22%0.23%0.22%0.22%0.21%0.2%0.23%0.18%0.21%0.21%0.23%0.16%0.28%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped