European Genome-Phenome Archive

File Quality

File InformationEGAF00001552386

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.

3 529 218508 989245 308182 877133 576112 36195 23083 70278 60171 94163 93962 57758 77054 09452 91551 44449 51749 91646 53646 33544 29442 80841 46738 85339 64637 98239 31336 94637 86635 69236 13235 50635 26534 15533 78832 56331 85331 85031 02329 86029 96928 76828 48728 40428 63426 62326 72526 42226 01125 74325 65424 43524 61324 45023 77223 70822 27622 60121 56021 21821 03120 44219 41618 99518 57918 56018 25517 41617 10516 57816 06716 10215 90715 04814 90714 38213 49713 65113 70912 82812 16412 08911 61611 12610 74710 60210 3849 8989 4019 1058 9048 5798 6858 1538 3657 5247 3636 8967 1576 7406 2236 1005 9335 8465 4615 3605 2104 8804 6884 3444 3524 3554 2463 6653 6713 6343 5183 5183 2083 5623 4923 1472 8862 8442 6202 6242 6022 7032 5132 6622 4542 0782 1362 2382 0842 1252 0461 9371 9331 7911 7311 6991 5881 6341 6151 6421 4011 2971 3691 4121 2031 3001 3371 1381 1851 0931 2021 0699791 1931 203983941899848857836811780800806847790719726791730665716791629618656720669698586464584559566586541646530569469439514428442438477459406393535446431479430389417428366393428330394532315367505365306363307450351341273296375296296303292438300295286272279258279277245277259254267319265289271238259333283262215217254285304336223372260264255252251253257241244273269289276262326283175210266227198214269201204206236229222181167190203303174181175331271156202172206200169187147174282282191161147161187187181166176153201190185206179193191192160251216157237149144153146165137123148140154202160130145134108129138127129135122112225151139145136126134134152108132124127113119124141110100919910911621410410010690110128966990967085908482711049093707680801491076370757168796188918420178811938088741868970762067172745781747077120109805716178866617614657605882845666154686170169575514593738113250162718454629568684843525237431443743623661456657404162465543564950534141465543403546483958433934394546524241383865556057708245145333042404327322833404133314846304236473840332936343666263331392457382323283335353030333835353828272226262621436726232421222721241726191319191020192125212413141915192919162123212218171320233032323129222731283922343229323326111291822211815201819191422111318182719262414111917202017188152012151414231012128158106101715151513201613183214118151071378111014121517512261578111511191215149169171215462912161212141613131014121481012991081315614101215151315109157121182117101616971114181111161281116109682871012613289109764246131410112428104811101310141298652911131049145810611710921041265101016645132394567858650897755121087810410810559613814121351129885210989532476561075347264912753141126116678913910681142385487165431012849786239145611632195965565611211784468555592544 751100200300400500600700800900>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.

230 255000000000000011 093 63400000001 611 02100007 588 7060000015 639 037000123 188 34700000510152025303540Phred 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).

97.2 %1 238 78397.2 %2.8 %

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.

95.7 %1 220 34295.7 %4.3 %

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%).

1.5 %18 4411.5 %98.5 %

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 %637 40450 %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.

71.2 %908 12271.2 %28.8 %

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.

6.6 %84 4216.6 %93.4 %

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.

285 5804 3452 3869 9951 8192 62916 3683 8442 6877 5752 2451 9848 4892 5691 2684 2811 2091 2355 4062 0071 3243 8961 6663 0486 3155 0661 47112 5321 2201 3014 6382 1111 3323 7541 5962 0514 3792 0499784 56825 8571 7315 0181 6572 3142 5244 2882 4939 81810 4611 7752 9281 4382 6975 1121 4611 3863 6182 5862 0971 067 756051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M# 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.

98.81%98.78%99.1%98.67%98.79%98.58%98.61%97.99%98.91%97.8%98.86%99.37%99.23%99.2%99.34%96.89%98.63%98.81%98.84%98.45%99.14%99.18%99.06%99.81%1.19%1.22%0.9%1.33%1.21%1.42%1.39%2.01%1.09%2.2%1.14%0.63%0.77%0.8%0.66%3.11%1.37%1.19%1.16%1.55%0.86%0.82%0.94%0.19%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped