European Genome-Phenome Archive

File Quality

File InformationEGAF00002445854

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.

761 025222 708109 30185 12168 41858 94352 37247 05742 75039 52537 57034 63430 85830 47228 49027 15326 42324 71023 59222 91123 00321 63520 83720 55119 96019 38018 97218 70918 05718 41318 35217 88117 17317 09216 61716 84216 67116 32816 20216 42516 00815 69115 67215 58115 29615 15315 48314 93414 97115 03214 57814 20613 91513 95014 11113 50012 84613 00312 63412 84212 97212 87712 72312 45612 29412 58512 51612 31611 97111 71011 56412 02311 56011 44811 65711 37511 46211 15811 01710 79410 61810 56710 66310 36710 49310 01210 0819 9609 7459 2789 2889 0769 2419 2439 0448 9118 8118 3858 2458 4248 0988 0528 1487 9287 6477 7187 5997 4147 3347 0566 9886 6906 6466 4826 3566 2846 0486 0495 8925 8395 8565 7215 7995 7525 3225 1965 3485 1695 1295 0514 9035 0474 9685 0064 6394 8164 5944 4684 4574 1984 0413 9653 8673 6373 8093 5963 4643 5613 5963 3813 2563 3203 2503 1473 2413 0513 1913 0023 0972 8732 9442 6972 7012 7212 6042 5782 5342 4672 5642 4922 4132 1552 1712 2492 1312 1612 2051 9301 9421 9032 0451 8861 8571 8611 7431 7221 6581 8021 7851 6531 6011 6161 5231 5381 4721 4871 5011 4061 4271 4311 2441 2591 1901 2231 2021 1231 1781 1071 1311 1281 1481 053952990954939906969920902969938777838894856740760730747781673690727670641657617562537567539568526562492516555472546424448431420424412466396421417358319367353381323337345274336332314296304274306274278264255271238223252274209208240219204206204216194191211179178204215170177157189158222158157178159146145163158163132146178157139163133148132150149144124137116135134158156139971071531151391121031121261341081269211498130107939813510710797919189152157978488937584877488627681826573717172717173686761727570626660556277604657565968586052455555584253475254385061586445474553465148505943384444573648304947393137183834424332424038263736454652352214283433373729292923442725232938273840302622233127323223323834223344272833293225163528243128192628152428303024202223252930311824252021252315222313181723161331131721111621202717202217131720162317231919182422271615332013181921239272724231615241917121813181321202213111715161881641712913151518101711151317121113914111611111210186619131516187151115161510101616111110121112191413221314251813171520713121013128111813101414510151314161015111451011171118241774201613212111151214141610920121511910176191925132212141681120121114812161115161916101623141410172015151116813101317148108181320161016675841710148118158145681323111210713121310129118613131514111012918201011161421171113121616614141010991011610767138139158141110129121410151311510141348713171712761114810710731110678111058715612511141010981281211107695191491117712139165887968461311161191511871239134686967296119585566123836338715361375746624512566332322361411855164765645661 612100200300400500600700800900>1000Coverage value1101001k10k100k# 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.

139 42600000000000005 230 6980000000239 72200005 069 7930000015 217 091000123 114 02000000510152025303540Phred 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).

96.9 %1 924 33396.9 %3.1 %

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.

96.7 %1 920 91496.7 %3.3 %

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%).

0.2 %3 4190.2 %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 %993 40550 %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.

84.4 %1 677 60684.4 %15.6 %

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.

7.9 %157 2057.9 %92.1 %

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.

130 2291 2784742 2916976021 4321 3215873 0621 2919513 4641 1026921 7674508021 2369944911 8911 2501 4222 5793 4348707 6699253 1831 8579497962 5454669951 2431 0228342 90037 8421 9341 3911 9344 2842 2612 1042 8942 7264 7044 3673 30616 6659394 2042 3511 0664 3662 7964861 720 912051015202530354045505560Phred 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.

99.84%99.84%99.84%99.82%99.78%99.8%99.83%99.82%99.82%99.84%99.84%99.88%99.86%99.84%99.8%99.77%99.8%99.86%99.87%99.87%99.87%99.63%99.7%99.9%0.16%0.16%0.16%0.18%0.22%0.2%0.17%0.18%0.18%0.16%0.16%0.12%0.14%0.16%0.2%0.23%0.2%0.14%0.13%0.13%0.13%0.37%0.3%0.1%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped