European Genome-Phenome Archive

File Quality

File InformationEGAF00002405891

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.

609 355 108319 383 726128 773 49860 204 13827 021 30914 049 5717 936 4925 150 2553 640 7462 787 1212 220 6191 840 8401 548 3561 330 3281 143 978998 498882 430778 750693 895620 538558 224505 100456 659416 187378 825347 764318 139294 806269 070249 846233 907218 357202 116190 357178 565166 845156 161147 935139 500132 720125 261119 218113 960107 696101 75397 68092 75488 92284 84981 74677 90474 88071 45068 68165 12062 78159 86457 46554 21652 39050 28748 43046 86144 79842 89041 35539 12237 81336 26934 40032 45731 65429 73429 59527 70327 06725 20824 45223 39322 30621 31520 42319 67318 43517 51616 83815 88115 25814 34413 78812 84112 51312 04611 24210 55910 0379 5199 1348 6428 0627 6037 3856 8246 6546 3606 2045 6835 7275 0744 9254 6034 5914 1844 1473 7733 6543 3833 3163 0692 9872 6232 6192 4692 4862 2332 2401 9811 9331 7921 7971 6731 7501 4581 4781 2781 3601 1381 2081 0691 1159869558398487628316848016256435867095535075745764555264245614184383623683244273393573173062283652473513103482492852332522123581912802532821762501892891822862131982182161691631492021591701661581281221142291161302031631112561201941171751251311201368511711419710315383206107196132156961698512011414086150125215851349787104128110106111168891048886125150135213127152638658118728660116647792997610691101981059569751136468706468856272681106673931245810385957064638974761081197788681537093856472152507298856670639074697194721018298801307210481917012067109641308613075128438951856386491035083577576137771277680701045510456537560587361806210270986393829054637879636464102678750106686064826710341655261626659736064705872605688768174794810758102787165865571547058764810057504573405149455965537153506578434638715255656745724955444440453993422337564847655234424842294533474061856255464884497345863663446761554350455249543570405245594066377526384535382728423638324129453137323627263430403927643638343633313139233539445776324028552944286629323430364229553834312827402233394953201833143017272747254221231424382932282538356627181939362523232136194523192226191818212512333320381422162320171917262016363551302116131918102224212115334021172625261616193121232015124220181535262249262213211326311316182071581217111717121026627151413331221131610121717171682311192112221316132120113416139221620342214162316271316910138111141218922193513202523620271515186812147108109201015112216371811101591313112313948141641951012518177102314311315718141413121511101961416172131613101597824122472413127961194152515766897710771138811141216155145205139108948541197124983356399512386435774553648102444289108649728614838694845267878122792211265841212636962024467656741149720 173100200300400500600700800900>1000Coverage value1101001k10k100k1M10M100M# 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.

001 556 14600000000071 602 23500000000035 248 182000046 844 3130000158 194 3200000287 884 6260002 366 631 12000510152025303540Phred quality score0G0.2G0.4G0.6G0.8G1G1.2G1.4G1.6G1.8G2G2.2G# 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).

100 %45 285 660100 %0 %

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.

100 %45 285 660100 %0 %

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 %00 %100 %

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 %22 642 83050 %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.

100 %45 285 660100 %0 %

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.

0 %00 %100 %

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.

80 204826 648124 32237 438656 10415 402257 4186 942214 114195 5282 344181 70892 892465 726318 504285 658306 188183 364255 240370 408752 5261 886 91237 770 070020406080100120140160180200220240Phred quality score5M10M15M20M25M30M35M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped