European Genome-Phenome Archive

File Quality

File InformationEGAF00001553476

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.

5 017 736953 650547 918408 943322 380272 895235 754225 555195 058174 070161 935149 047140 368132 325126 979120 726115 035108 053103 13799 89995 04893 99488 48185 29183 94682 04280 75477 30476 31272 27572 48166 97866 01063 90063 02462 38859 17358 28956 44456 68352 84951 66951 36450 58449 38147 20645 07143 99543 58241 45941 98440 49440 07438 89037 62136 19735 69334 75733 16733 24132 35931 28329 72927 93527 47626 01925 39324 71824 47323 26022 30422 05821 13020 96920 01619 61619 28018 23217 61617 03916 81716 21016 24015 11214 88413 89113 45813 17012 35212 15811 58311 27010 80310 3029 7049 6889 2928 7978 0898 4197 9897 3697 4967 1996 7886 8796 5126 0985 7875 6475 5405 2234 9734 6754 6774 1804 4384 0943 7743 5803 6313 5683 3343 3783 2913 0513 0413 0443 0172 9022 8902 7012 5282 5222 4252 3882 3782 2662 2122 1091 9411 9771 8251 9341 8301 6181 8271 7161 6531 7391 5931 4481 4931 4151 3351 4691 3921 2721 2861 3081 1901 1951 2201 0781 1361 1341 0651 0951 0549951 0329581 1389458898678338387967637538489218999288958358437677507806506866566306976396546385946955905684894915405285775454785346085114385715195166175294254214534464644474664383763693463373884023663533443483303303263125063323263152883063692723233353252462712703382542352123082362742112371992582112061872163082822302291751672101671993151892721481771863751341702311531571721432882361691601572201881792771801451251541841731881781671721761821543611221491881651531371791621551241221201282121362232441018510832393118117105112150115191117939611510710470727176107208761921168311098911829294971349076711938060717469191217686511383109909372891108787671201128772787672844664811737567641742586072627370855856598271786567676349695363596461565857735251561665479128575758574946433345139652946492953583936365444423839411355337433930615342324721253420362829694336342627312934192250128273033242620201923302441362536325714631293731344014234403633182923703031282233312726222024241502727182940192631211217231318202924141622172021178018124012815120153914101915122915133721153515131616352021151718131820134121514241419141318231920168451715351413171424201119991210131115666125915101112199781141414101614101511614121325106163181411912121613308141013223818101811141712323835286789121014197910611510712111111837132491714931413781012106951061411114786131081525128313128111215132212910913813988114147123415121318167552731195111096607812121122108614516615111411715118588131368510815998895799121031754108758447813614412106936767714481581211312813533244361111891276893658555966855126101081213128481091115121472516131261711810781568391084579786555575432543337948345256421346587676353155422644147538717 061100200300400500600700800900>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.

561 744000000000000015 448 49300000002 600 979000011 406 4330000025 297 746000223 427 60500000510152025303540Phred quality score0M20M40M60M80M100M120M140M160M180M200M220M# 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).

94 %2 096 57794 %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.

91.4 %2 037 80691.4 %8.6 %

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%).

2.8 %58 7712.8 %97.2 %

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 %1 114 97250 %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.

71.7 %1 598 40271.7 %28.3 %

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.

15 %335 21615 %85 %

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.

813 9566 6422 98114 9332 2263 16836 6875 0632 8759 5382 5992 62413 5303 2321 3405 0051 4691 6428 1222 3961 2915 0572 3124 0538 8076 2771 66014 8281 6971 6816 8012 4531 6364 8691 9962 7306 4262 7751 3505 84032 8382 3626 9382 0422 9563 3355 7693 21211 20111 6052 0093 1752 0003 0237 1261 4791 7613 8393 2332 7641 586 010051015202530354045505560Phred 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.

97.32%97.77%97.58%96.93%97.42%97.96%97.82%96.64%97.38%97%97.76%98.33%97.37%98.18%98.09%96.53%98.08%96.08%97.13%96.99%99.14%97.88%90.1%99.69%2.68%2.23%2.42%3.07%2.58%2.04%2.18%3.36%2.62%3%2.24%1.67%2.63%1.82%1.91%3.47%1.92%3.92%2.87%3.01%0.86%2.12%9.9%0.31%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped