European Genome-Phenome Archive

File Quality

File InformationEGAF00002485704

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.

442 158227 86088 66884 48061 09558 19349 98947 03442 56142 53838 82737 87836 68434 66932 03832 23929 95929 94429 19627 73126 95826 99225 85325 08824 08924 13122 76422 95421 96022 21220 58820 57020 04419 88719 19018 80718 05517 34617 27116 53716 37416 16615 31415 47614 38614 26913 56113 56312 61212 38711 98111 67711 21610 73810 42110 3409 7739 1829 2668 7658 4098 2217 6647 5477 5337 2856 8676 6856 4126 1346 0935 9115 8105 4095 1455 2654 7254 6564 6784 5464 3424 2093 9153 7183 8253 5373 5793 4593 3093 0412 9812 8922 7892 5652 5152 8382 6072 5352 3372 2922 2952 1231 9042 0001 9161 8351 8651 8791 6051 7521 6571 5581 6911 5511 4921 4021 3101 4001 3281 2191 2261 0591 0891 0169821 06594085894996279890876389775790471377669257558572260972461670961753752559064663250247846547940951541448533743037850244650838436129829533431428936832738229829626541328128123640826923924522924825526722528525823824624423521723020816623929518727521219326928118619518518019613319816019223215819518914918218517330326516218818616015516616717614915115616112813920011913310312912013315314716313913113613716512814111911912219410016511713810018312711915811511210710219411718310217212417996991071059311710711511810310711813612310112312011612411612812412111510924812210117211710084849610287819210565798783978096861598776958987869194901481077183596879697376736165551315168646653466252455162565551471225438455355535359454849506167436543117473037733850284731432327385233444146303134423435444539323641463937102322129332435434432283630293844352638441073443402129332237243235343333302926373135292127282933252826273522233535291833272118283029252114262422272124273026162325222313242322932122342123381721311724172017142114191918141923151823152022201613181619101213182320817151431201319181513211611162111121010111710816151192017171515119412510138214159111011146169981612131513152791412141391114198121113101412818111061918141112122023121720171014251920231413231711151617201219161812101321243417201519262212142515172316181618221418131616121814171522617182415221258918152819162017218106161220209121120851717177176201316121312112519171517121921191720162215972023181721162519179192118181723162827242616161434181618151919272716172422232021191320292125202319222316312837303516292419182320252017282026252720242734262626272020283326323820302125322620292829172737272024212528312327322627252022233029202621142323221721281517141332142517231423292219232424212923203019131521242812241524181719261819161933282322171823232421151917222023162228381923162224251017232332353036282720302726192225223132242828282826341022124392432272020213230302124202119281424202417186 091100200300400500600700800900>1000Coverage value10201002001k2k10k20k100k200k# 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.

14 20300000000000003 532 6530000000243 32700003 722 8640000013 071 775000115 608 27800000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M110M# 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).

58.3 %1 057 99258.3 %41.7 %

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.

58.1 %1 055 64658.1 %41.9 %

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 %2 3460.2 %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 95450 %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.

49.7 %901 72049.7 %50.3 %

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.

7.3 %132 5997.3 %92.7 %

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.

969 8472 5056073 8191 7071 0572 0273 1888506 7461 4611 4714 4902 1678123 2326888741 3582 0234223 5791 3732 2013 4965 73781412 4219213 3151 4551 3636063 4054128381 0571 8406293 97640 6611 4571 1061 7893 0742 3352 7291 6721 8993 3692 8533 04312 5808472 6581 7445293 9072 070432692 783051015202530354045505560Phred quality score100k200k300k400k500k600k700k800k900k# 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.

99.73%99.75%99.8%99.77%99.65%99.86%99.83%99.64%99.8%99.72%99.91%99.88%99.69%99.83%99.86%99.76%99.7%99.75%99.74%99.88%99.78%99.68%95.46%99.96%0.27%0.25%0.2%0.23%0.35%0.14%0.17%0.36%0.2%0.28%0.09%0.12%0.31%0.17%0.14%0.24%0.3%0.25%0.26%0.12%0.22%0.32%4.54%0.04%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped