European Genome-Phenome Archive

File Quality

File InformationEGAF00002485288

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.

583 824244 50572 15169 15438 16843 47724 71226 85318 74519 71115 87617 13713 33213 04612 49012 23410 42610 89510 30310 0519 2799 6799 2769 2788 9018 6978 3518 0458 2508 1037 9287 4967 5537 6847 3017 4926 9137 1707 1596 6426 3426 3346 6696 6476 6326 6385 7305 7936 0446 1756 0655 8695 7756 0825 8485 7365 8605 6605 5255 5555 5575 4395 4845 2614 9855 3685 3535 1905 0605 2334 9504 9474 9454 9985 1724 9334 9555 0014 6834 9195 0504 8124 7344 7024 8594 9224 8574 8704 6914 9504 6324 4244 7514 8764 7234 5514 5424 5274 3774 3954 3674 5874 2834 2304 1754 3974 0644 2094 1744 1854 2124 0594 2784 1793 9654 1083 9394 0063 8023 8513 8423 8793 8973 9373 6733 8633 7463 7043 6553 6953 8703 7073 6333 7493 5653 7153 7223 4493 5063 4193 2873 4343 4843 3583 2793 3043 2783 2993 2523 2943 3503 0663 1243 1003 1382 9763 0702 9032 7802 8643 1852 8893 0053 1182 7462 9422 9852 9392 9262 7582 8282 7662 6852 7282 7302 6632 7712 4892 5642 4962 3802 3152 2272 2932 5202 3632 3512 2132 2422 1972 1142 2402 0682 0792 1432 0642 0472 1122 0151 9551 9451 9081 9371 8871 9521 8781 8821 9091 7621 7211 8101 7901 6201 6161 6421 5811 5341 5601 5711 5751 4991 4721 6171 4261 4421 3771 4021 3461 2581 2811 2681 3061 3291 2801 2211 2041 2801 2721 3461 1981 1781 1921 1501 2441 1101 0209971 0371 0231 03592996098399091788188199790087391887396784889375187579082076374870570672468070563966462663658665563361464160759853755259357954054650949646950648254250645639945842844539637140742939537739134432035034634434531538935033828430627829138831129124529124327123724627423023424228124029126323919121420222721215019120418018717116918715817719117115415916816517316115517415314617915817916416016715317115616916614315511711312113514612013813112017211515310699118102109131108901088385908486909075796684818687939795100791098210510710891758597123761037093959010010696616576551354253746985826058485963808766594964656070616476796663585146535048505342645061454162464247533947504546455849503350334046463360534851394559413855405142486042283929434337404052393438443634452237542838415740333941384340552833482543394239332929314135363131244227402249302433382037382629152332282730262728212230235222362327191528432829372824212921552326392024261742253525202723242221251818211813311721183221301718172419251426232120221312111825212591520222511192317201825212828142215172324182323172321142416818151726141424231820251917161518261615151928312761182412191419162216162219122622282717171625123217151819741915171912715715221415121772192115171511232112171115111612141917161511111214119125111515812109141719581514161215171424881096161111171186912151371781181115815131311515119879611811971121175101181281014158611986117181412136121168668106910122869131191041261315746436728576126138665534657681085310783105359988638101043547957455834245794461065976336575968711667910585 652100200300400500600700800900>1000Coverage value210201002001k2k10k20k100k200k# 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.

169 17800000000000002 716 1630000000147 00900002 641 3280000012 394 075000122 909 44700000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M110M120M# 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).

93.8 %1 762 47393.8 %6.2 %

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.

93.4 %1 755 99693.4 %6.6 %

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%).

0.4 %6 4770.4 %99.6 %

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 %939 84850 %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.

76.7 %1 441 38876.7 %23.3 %

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.

11.5 %216 95211.5 %88.5 %

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.

208 3313 3571 1235 7311 5691 3823 1514 0831 1437 5562 2932 2407 3022 7651 4035 4811 0041 6032 4382 6479855 5862 2393 1595 8648 9671 33319 2261 5675 0302 5422 4031 1125 3687001 5271 9962 9401 2217 01557 2792 9302 0082 9305 7883 6982 9553 2233 2456 0635 1365 57824 7611 7095 0624 1471 3707 3844 1628481 437 627051015202530354045505560Phred quality score0.2M0.4M0.6M0.8M1M1.2M1.4M# 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.

99.76%99.69%99.73%99.73%99.62%99.76%99.73%99.47%99.73%99.68%99.83%99.8%99.72%99.54%99.81%99.75%99.7%99.67%99.58%99.62%97.43%99.33%95.57%99.9%0.24%0.31%0.27%0.27%0.38%0.24%0.27%0.53%0.27%0.32%0.17%0.2%0.28%0.46%0.19%0.25%0.3%0.33%0.42%0.38%2.57%0.67%4.43%0.1%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped