European Genome-Phenome Archive

File Quality

File InformationEGAF00002445540

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.

533 724128 42355 40442 53229 82523 98819 63817 38414 84513 63912 22411 85911 21210 3509 1819 2458 4917 8797 6707 3427 2056 8186 4966 7316 5016 1466 1025 7055 9206 0106 2905 6745 8395 3445 4915 7535 2794 8534 8634 5934 8254 8854 8225 1274 6434 7614 7554 8614 4714 8564 4264 6204 4424 6684 4494 6294 3074 2804 2144 3424 2884 5394 3094 2894 1904 1644 4724 0844 3044 2544 3114 2904 4784 0024 1934 1944 2014 1924 2674 2054 4404 1654 2384 1084 0444 1634 0673 9243 9794 0923 8423 8773 7673 9243 7683 9313 7963 6924 0633 5453 6723 5633 5673 5913 6873 6163 7463 6253 7133 3643 4403 3543 6203 6203 5133 2733 4443 3453 3453 4523 2643 1423 2693 1963 2443 2483 3693 3003 2483 1513 2113 1963 1853 0213 1312 9552 9842 9122 9873 0712 8922 9922 8652 7992 8312 7332 6322 6252 6212 6522 5262 6362 7372 5922 4542 7102 5532 5312 5472 4842 4962 4982 4212 5092 4472 4292 4382 1932 4252 2302 2012 3732 2952 1822 2172 1742 2662 0291 9642 0282 0042 0901 9791 9301 9301 9271 8461 8821 9531 7831 7471 6571 5681 6871 7321 6041 5881 4841 5581 5621 5121 5001 4231 4081 5031 4541 3891 4461 4281 4481 4331 3451 3521 3381 2631 2671 2521 1791 2491 2541 3211 2231 1401 1401 1541 0891 0851 0221 0081 1061 0511 0909549929919509089258579249289438198519118288608838777838068658027868247777076917137386816626566306226075976086396205996106555505815985496005935435495145064785234794274644714974264254214043663684163603953803543663463493203472923303233292963282932662692702282482942672592492592352332762182262052141961972131781961772142041781291761381431471481441371551551861071371281211361241481081051049497949689951208511310611080889790958012284818077738669678993717777758266697010975837359906058756287455365816372516768547955525446483459515747334350405654524526283734373343374310340363845543446443940363544403576332333313044203231423224373629262231152623331945192619192119212221202325202624192032222825171920242521222023191426192831312910242224122122181517252343161722292127142219182212201913141924211715181728141618971710162312131620111211131015221325151518218171516111110161318132017121215176181196139815812578106101212101513811786171081511127985118118710107131157649119131012995195551063844551531051191277911858412106124131014113891181333514910771012710451097715157378891881112155310768791349128128711131461310141410109112767959611971056958737119156510476753551135434553131166910271010397715533136775531113577463648910896797353512654114986626767569674463311624466359777108751141333627101057265844291236253157169155773236523175354291454313433516443635237236674516466353144133313 762100200300400500600700800900>1000Coverage value1210201002001k2k10k20k100k200k# 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.

18 43000000000000003 760 0450000000194 49900003 496 5760000010 671 93600084 003 11400000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M# 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).

98.8 %1 345 56698.8 %1.2 %

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.

98.7 %1 343 72298.7 %1.3 %

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 %1 8440.1 %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 %680 96450 %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.

79.8 %1 087 45079.8 %20.2 %

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.

12 %163 13012 %88 %

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.

98 4482 1265773 7168501 1121 9572 2788264 7131 5731 5824 5471 9308413 3025961 0381 5721 6053893 4921 1891 6913 5085 12585716 6701 1223 5421 6271 4968334 3254298981 4081 7499515 38438 7051 6431 2681 7403 7942 4751 6172 0172 3674 1593 6604 31119 1598583 6312 2069067 3462 7534671 091 317051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M# 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.

99.88%99.89%99.9%99.92%99.92%99.9%99.88%99.84%99.86%99.9%99.92%99.91%99.91%99.93%99.87%99.87%99.89%99.86%99.68%99.89%98.91%99.77%99.3%99.96%0.12%0.11%0.1%0.08%0.08%0.1%0.12%0.16%0.14%0.1%0.08%0.09%0.09%0.07%0.13%0.13%0.11%0.14%0.32%0.11%1.09%0.23%0.7%0.04%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped