European Genome-Phenome Archive

File Quality

File InformationEGAF00002484816

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.

479 076183 61296 96577 59662 63953 23547 30642 14938 56536 61733 28931 74430 09328 51826 84726 41325 61725 13224 19423 77822 94722 38521 24521 47521 44720 21220 74418 81218 89018 76918 34417 91617 83017 15516 77017 39916 77316 75116 95016 93216 53316 08416 20315 71415 74315 20514 85015 38315 01314 65914 50014 43213 84313 51713 57913 20312 96713 06012 75812 18812 58512 46312 35111 71411 21011 40310 95711 02911 28510 44410 06310 3919 87810 2389 5459 4199 2579 1518 7378 9188 4758 5118 0147 7477 6787 1207 2947 3887 0887 1196 4476 6966 5726 6666 1275 8336 1125 9295 8085 8665 8755 4205 3095 1885 2365 3235 1654 9574 8794 7044 5784 6434 6774 4234 2934 1354 2253 8604 1913 8573 8033 8543 5563 6033 3403 3653 3753 3023 1893 0093 1682 9552 9482 7622 7062 5532 6722 4392 4862 3102 2532 3502 1472 1552 1122 1751 8551 8701 9251 7521 7131 6551 6971 6411 5841 5951 6981 5801 4761 5191 4711 4101 3851 4071 3341 2111 2891 2871 1651 1471 1341 1611 1141 0309759769538488299049488369238878418068028007467186917236926606105965946247076107335415625825105345094904514863783873914143993894453383483912983653533243053102862973083173103023203192912842602853453222682742492551792692322172352181971871941791792041561811971722352291691882031701861501381701801881941511421511741441441441401831781501901211501231291681861201321611411231299995134103135109126110129122109101111119115949788102948699100979394781039697998894807986891137992708610668728884838480778490114616897797713386666690666379687670907082103766274737282598162647193595970687486756770456762665684101645766786065635248505962455468675077624361496380684867676063686268119844863546749696480575858795451565864547670675371926854765865625450628368636555476871556358584047625951615449525768446266597439476565556065585157455248524436554955473943425548394742484046394643445337354448493640494041543547334949474552533657523949395044334134383837394538394551455051474745454352665149515932484258684064634963445348415761527352645758485153595549523946465057634239445764466049464246644549695946454841425148484251474551515136425255616559563953515146614253404376423144494451485057345156534056524646494557386044535954514946583554313952424549404057495648535342454551465554394143364740404626413627304342513843433234395228503541524547503832334423383933413352384333423734374935464728364740413126313346244025333634313026332839323229313330312038332521253020242925273231302718332727292724272621132428232620262917242320142121253123182822171728222321293825262221373727312314252035141714273022211915182220201921161922121616131716161318151917161310913111613171511121325757912891316171015111818111011121713131615813207811121511111221871077129981391581611141712145767689149766128138108413512117133974 755100200300400500600700800900>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.

3 10800000000000005 033 6130000000313 07800004 960 8190000012 495 79200099 327 19000000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M# 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).

98.4 %1 602 20998.4 %1.6 %

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.

98.3 %1 600 51898.3 %1.7 %

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 %1 6910.1 %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 %814 22450 %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.

79.4 %1 293 57279.4 %20.6 %

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.

10.6 %173 03110.6 %89.4 %

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.

213 0282 9206515 7701 0911 3492 7263 6518246 8282 0902 0907 0782 8731 2644 8189431 5062 1372 7224834 5512 0483 1125 0067 6091 22722 9451 4466 0021 9822 1341 2165 3226721 7341 9582 8201 0986 91748 4202 3121 7732 6345 6743 4092 2192 8872 8235 3934 1306 36421 9921 3964 6333 2761 0338 2433 7266341 191 362051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M1.1M# 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.

99.89%99.91%99.91%99.87%99.9%99.92%99.82%99.9%99.9%99.92%99.93%99.89%99.89%99.93%99.92%99.71%99.91%99.92%99.9%99.91%99.88%99.81%97.81%99.96%0.11%0.09%0.09%0.13%0.1%0.08%0.18%0.1%0.1%0.08%0.07%0.11%0.11%0.07%0.08%0.29%0.09%0.08%0.1%0.09%0.12%0.19%2.19%0.04%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped