European Genome-Phenome Archive

File Quality

File InformationEGAF00000655946

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.

82 658 00137 674 30413 428 9646 304 7372 592 1011 704 616924 693697 791463 181360 881273 487219 634175 714146 025120 499104 58689 73978 72969 46261 88855 45249 74345 02341 61837 84234 99031 71029 69427 53326 53324 47423 09822 03421 25020 17819 63018 56917 80416 94316 83016 01115 31415 28614 61414 07213 96413 47513 04012 82512 44012 25612 18211 74811 58211 50811 23311 15810 91010 80810 26210 39810 1289 6679 7029 7249 2809 2018 8739 0058 8428 7778 7218 6358 4828 3798 3578 1598 2078 0998 0767 9467 7057 8087 6617 7567 3457 3637 3407 2427 3447 0826 9546 9887 1777 0147 2116 8766 7606 7666 6976 6826 6356 5046 5136 4346 4026 2316 1506 0745 9405 9895 9766 0755 9056 0106 0745 6585 7165 8415 8105 6505 5435 6195 5675 6145 4965 4565 4935 5015 4895 2725 4195 4605 3255 3405 3585 2905 1755 2415 1385 2355 0615 0565 0334 9144 8274 8934 8654 8224 8004 7564 5944 7284 7894 7004 7324 6314 6134 5424 5984 6504 5234 5904 3094 4104 3024 4234 3364 3434 4594 2554 2624 3744 3584 2584 3374 2774 1994 2824 2094 0954 1624 2244 2174 1424 1234 1194 0074 0074 0654 0664 0494 0714 0524 0563 9814 0524 0193 8913 9633 9513 7763 8823 6923 7683 7903 7733 7493 7353 7223 6653 6663 4793 4973 5933 4423 5823 5163 5113 5913 4933 5373 5183 4923 5583 2673 3253 4753 2333 2343 2613 3313 3383 3103 3843 1313 3463 2833 2413 2783 1903 1153 1913 0223 1113 1823 1093 1573 0813 1093 0873 0332 9083 0683 0273 1302 9762 9222 8363 0012 9532 7232 8392 8562 7542 8222 7522 8362 7142 7382 6652 6762 6742 5852 6002 6432 5272 5012 6402 4972 5392 6072 5762 6442 6442 5912 5502 5362 5242 5992 4562 4862 5252 3822 5212 3962 3842 3202 3152 3722 3382 3372 3582 3302 3332 2592 3862 2142 3142 1642 2082 1852 2152 1532 1842 0982 1482 0432 0621 9911 9822 0421 9482 0821 8211 8811 9681 8691 9121 9051 8251 9101 8301 7671 7581 7901 8031 7691 7651 7761 6741 7331 6091 6351 6511 6361 6361 6101 6011 5991 5901 5661 4961 4581 5601 5211 5161 5081 5201 4531 4431 4751 4141 4751 4021 5111 4021 4531 4361 3871 3941 4041 3721 3921 4041 3551 3411 3141 2621 3231 2491 2681 1961 2891 2531 2251 1811 1621 1991 1201 1531 1791 1111 1631 1181 1771 1491 0831 1581 0741 0961 1411 0591 0641 0801 0931 0481 0061 0561 1081 0721 0491 0421 0761 0551 0591 0521 0081 0071 0049819288899489821 02997797995095592388395391190287686692087785284286883187289383483183282182881182477782874375471877071772970872876468968074475272569573368271470471465968074366668969565865165662764162461860661762361860157759156559057955959557061253456660355150054452849747051242650551545247745646945343145445945342847241547041441442843339641739239441341242342937236037937336032034230035033836032935533832030430930730132428628029827328729928229229326128329127527428326023823725921720220922817220620922422419422021020319920817118618217619715217420016420416417318217817316919615315814116615216115015414813914914215413513614313913014012114411813011211112115212411188981091011209790899192105839698808682857879657581836981806972786668697375666258715366735363584358605161515954465645415950535159505361474543464039444843445247294442484840492045483746434238436248424640504649394241533929304031394038303631282828253625223229333434493535323129332732342520292619242320222516241724132018102014121823131618151715151617171517141622181815122418201917121717162326212022201826201920172222222518181716251721182025161715208282818152523201127231320181724193015141715232516211517162114162017121817201617171314152114914159101620191819131210191411191314915182117141911181622151813813231823101120181313151212181716121316171710102115111010151011128691271081111131375874910759129107139129134 154100200300400500600700800900>1000Coverage value101001k10k100k1M10M# 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.

168 1520000687 209396 0592 179 6901 699 841609 6101 034 345391 663412 636777 511313 793946 329759 469867 5041 549 504843 1941 326 381858 5331 583 9042 214 2692 390 9072 700 1014 390 0624 376 9244 011 3054 409 0869 617 44815 401 0739 594 01116 409 95831 713 06043 876 69223 309 51360 599 96443 533 05871 931 74285 490 895170 218 25500510152025303540Phred quality score0M20M40M60M80M100M120M140M160M# 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).

99.1 %8 238 80999.1 %0.9 %

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.

98.8 %8 214 95898.8 %1.2 %

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 %23 8510.3 %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 %4 157 29150 %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.

98.4 %8 183 95098.4 %1.6 %

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.

29.1 %2 415 40429.1 %70.9 %

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.

363 6561 4126912 2101 0271 2331 0861 8931 66310 0936 7043 14312 1353 1912 31736 1393 72312 96411 5593 41120 38476419 03368 9776433 830519465489279 4281 0299509321 2209561 68838 813143 2664 5061 4525 1764 3701 1248 8602 0522 80833 5804 8285 1466 21410 5424 73213 75410 53415 11427 03649 5747 039 544051015202530354045505560Phred quality score0.5M1M1.5M2M2.5M3M3.5M4M4.5M5M5.5M6M6.5M7M# 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