European Genome-Phenome Archive

File Quality

File InformationEGAF00002484292

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.

688 436215 976121 73298 31578 98773 36265 97160 89257 34154 90652 79151 20448 26847 62245 88344 39343 39242 65342 41139 68340 24139 89239 62038 36638 05737 77237 49037 81636 27836 94937 05135 83136 16536 35936 05435 44434 55534 79733 48733 36133 37732 34532 52431 81531 20429 83229 50728 44127 95128 26827 37426 81426 02525 87425 36824 21623 88223 37723 01722 47221 71721 28720 50820 25019 76419 24518 90418 78017 99517 29916 55716 84315 63015 16014 92815 03113 75813 65013 41612 82012 41111 93711 58011 49511 17310 79110 2979 9149 2729 1839 0778 3878 0948 0498 0617 5787 4716 9186 8246 6256 4745 9806 0215 7965 4345 4015 1724 9924 7194 4904 3614 2994 1143 9903 8043 7333 7373 5913 4503 3433 2533 0993 1692 9302 8232 7062 4902 3692 2002 4152 4442 0872 1512 0702 0111 9501 7271 7631 8471 7881 6351 5341 4941 5531 4271 3441 3391 3341 3221 2771 1631 1941 2081 1461 01598298199399798190488691992092490392188874180469368572771162768565162066559258667361061556253451856653052052548045845844243443546544438439545238943235736637138737031035533134633436430632239331629928025628833428926428728830830236225023128122122522023921922620419618725024220322728219622622624026220921523321419420823419720918822418619421920221122019419820116618918216421321117120315516222417117316621617815017120017515816618216117614316116215215716014514817317714613317516819213012212813111312313814111612311310913413213412912193104110109112107118909011087931021051061029699948991114847093961568886100758193997610386661031331286463639093716463996184746788160678787761348472701019680102110123949075866687937684837681736870738361711136182688073605971737248445355696011769113465766536765515842585650635142505745643555615466686347656369528157756275505764746950645264624959676910067545757627556526663716771616645656769545680598153604965625039595657584866556450526462525541577842526361445358595857505860666742544155536956478246604054484847425738424660100423446313836532553545239387930442744383846451034747303648406250555746404747434636454838593748483941497054528045515453494651435765584350474934504351485367614551495857496072445162544854525145625846494236395139504544484039343949464948804538434533414538333839364235345235342938283439483426395023272622421833364023343632313621323518342828403826212817232235202539413736283234263014382020393025232319161821252358232222352422242121232216222427252119213224331824193026242919152726191823216148111420201712251516181914181217101623121411111319141811818111713121191711814111681023131113168791313915912812778588228108101314591189211148991414874476713978181191617794510499451277171451169131397158787610615117115117125111051014116103696987697115710769129958713106947159811114 192100200300400500600700800900>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.

120 95500000000000005 780 2140000000311 48300005 779 6310000016 702 602000126 178 91500000510152025303540Phred 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).

99.4 %2 052 10199.4 %0.6 %

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 %2 049 79499.3 %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%).

0.1 %2 3070.1 %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 %1 032 49250 %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.

79.6 %1 644 58079.6 %20.4 %

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.

10.1 %208 62310.1 %89.9 %

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.

182 6722 2377124 5299159692 3852 7138465 7271 9222 1106 1882 3771 2503 7787431 1531 8281 9165073 7521 2022 3363 9716 2411 29020 1791 6344 7562 3311 6111 1434 7096881 3861 9212 3251 1265 58649 2062 3162 0072 5575 2983 1052 1932 8694 3005 3294 5436 22621 4491 4775 3463 4531 2098 9004 0378611 691 133051015202530354045505560Phred quality score0.2M0.4M0.6M0.8M1M1.2M1.4M1.6M# 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.

99.87%99.91%99.91%99.88%99.9%99.85%99.89%99.91%99.89%99.89%99.9%99.89%99.91%99.91%99.9%99.89%99.91%99.91%99.86%99.9%99.88%99.82%99.76%99.92%0.13%0.09%0.09%0.12%0.1%0.15%0.11%0.09%0.11%0.11%0.1%0.11%0.09%0.09%0.1%0.11%0.09%0.09%0.14%0.1%0.12%0.18%0.24%0.08%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped