European Genome-Phenome Archive

File Quality

File InformationEGAF00002445899

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.

1 057 781317 755128 03183 67959 41752 19545 16342 00138 21235 74131 02328 67328 25626 93426 06526 54424 23523 87322 42822 96421 66321 28020 94820 07319 76319 84919 21619 25218 91919 39918 44819 03018 95618 14517 88717 82117 41517 63317 63717 62217 81117 03717 19217 29417 23116 87216 44816 46416 32616 21015 86015 80615 16915 24114 95414 87314 74914 48614 48914 13113 91814 55314 22013 53613 21513 05212 75112 79512 30812 47612 37812 49511 89811 97711 55811 71511 58711 17010 88410 92611 07510 73510 73610 4489 87810 02610 0499 5939 2309 0869 0958 4578 3828 3508 0688 0077 9487 6217 5077 3637 2387 1346 9646 8726 6266 6076 3966 3355 9565 6665 8395 4215 3915 2595 1454 9524 8074 6174 7764 4864 4034 3604 1524 0763 8793 8903 6673 6883 4493 2983 4103 2413 0192 8952 8852 7962 7132 6252 6102 4612 4422 4992 3322 3702 1832 1582 0832 1172 0181 9451 8591 8501 7901 7301 6761 6331 5421 4251 4401 3651 3781 2711 3301 2301 1441 1361 1481 1401 1391 0549811 0181 0171 0419609818648728737808017897727536917606266605497786086325965935526025335726245254614784294304005224354133673923774153723773673643633473713153464413653132702822423372903672672342532852622342522692512402552552373112332462592092052252332941911771981971702321921842042001792021952131972212292011841991852111991733001851671862091741731632211461571541791591641201361181231351301411281241111231081161181411281811191011021161131261111391341161118090105101949010310394108117101909111781979387100124859686921151051109986101639672751068472748286727671639276635550648380976867976670626160585645617153484513667536350545055515945556238464953505951494249574943424344485667329842433834364044461035151404154344229243940273230353420303828254537353630323224332540332221243140403627401720333233304026273343152020161735293236282324231825271621321826162459281823182618192415172514182213202119251921311720211816162916121823271618252314221414127121613141621241715141326251820161618121223161716121816191213201517101611231114161718121414201510271011141791812151272215141813211191421101315172416141212182111181171517168612871810694118561013911151366987781039147567610910554710710416373104655145841563912101067101743991012111011156879710910558747763991712511101610599131166551061087118264108684426810510379454445118544597813187136788355274466866657748584787751011737757437366246510559245875557736567793442471168101034355334401214646643324122522284646864358754423574666346341151117535346651431632273321242326321111331233223252132322 251100200300400500600700800900>1000Coverage value1101001k10k100k1M# 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.

6 52400000000000005 900 6340000000296 11500005 647 2410000014 789 961000109 410 42500000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M# 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).

89.2 %1 617 38189.2 %10.8 %

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.

89 %1 613 81289 %11 %

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 %3 5690.2 %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 %907 00650 %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.

71.5 %1 297 76871.5 %28.5 %

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.

11.6 %210 73411.6 %88.4 %

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.

277 4712 1495763 9341 3519602 1792 6949355 3801 9591 7486 4092 2041 1413 2396851 1041 6031 8504503 4511 5822 2333 6395 8171 13915 9611 2104 8181 8511 7221 0224 0416641 2681 4531 8731 0494 70341 8222 0491 5242 1134 8252 7532 1392 5062 5244 9094 1264 47119 4099984 4982 8368805 9223 0656031 357 983051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M1.1M1.2M1.3M# 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%99.8%99.79%99.68%99.74%99.85%99.83%99.75%99.59%99.76%99.87%99.82%99.76%99.84%99.83%99.76%99.76%99.82%99.83%99.85%99.85%99.39%96.72%99.92%0.22%0.2%0.21%0.32%0.26%0.15%0.17%0.25%0.41%0.24%0.13%0.18%0.24%0.16%0.17%0.24%0.24%0.18%0.17%0.15%0.15%0.61%3.28%0.08%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped