European Genome-Phenome Archive

File Quality

File InformationEGAF00002339187

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.

870 075 767663 402 486380 753 057181 132 57775 267 56628 478 95510 217 0603 671 0031 448 862678 713399 965273 335206 732163 622135 276114 77897 93187 90477 21468 53862 48956 75652 06947 96243 26341 57237 43835 36433 45530 96229 87928 34927 02325 00923 73923 21821 61220 51120 14018 96018 11716 62115 66815 27814 58413 94813 26412 85512 42811 96311 36011 08910 76810 04910 0579 6189 6659 1038 9168 4098 0357 8267 7467 3297 2236 9066 6886 7546 5856 5466 3496 3806 2035 9505 8495 9435 6335 6645 3785 4875 4495 1634 7204 9614 8104 6124 4694 3424 3024 2904 3184 5294 0683 9023 9603 7233 9853 9073 9493 9954 1583 8443 8043 6273 4213 1323 2313 2493 2383 1473 0623 1273 1672 8723 0042 8903 0582 9242 8852 8712 8092 6432 8372 5742 4172 4432 3712 3802 3652 2172 1942 2612 3242 2032 2412 0682 1352 1542 1212 1311 9962 0681 8621 9081 8621 9661 8651 7491 8211 8161 8141 9821 7831 7171 7201 7481 7451 8051 7051 5771 5861 6201 6211 6181 6401 6741 5871 7991 6141 5741 5071 5541 4891 4441 3901 4051 3841 4521 4291 4421 4431 4551 3591 3911 3381 3281 2681 2051 2121 1271 1981 2091 1561 2061 1341 1511 1011 0371 0601 0481 0871 0851 0591 0039249471 0089379239659519899879848918748818628718929588388898058097908038236977907547957146937496797287557356776636856306706606336065986226836436065686225996095736776596617086316546687216828097146626956745986226677246716507507166376526566776316505586296176436806385906295935306075686335765886095405865815375545965935605916066476226326626225976065916036126406306276185515746146085825926316636846315986356556286216466806866586316097016786506476346616716996536746836636446246496606076686456566416456776276296586847026906906326456446256386346876756326696766506626236746686266566416165986316205966296606496566876406356316365886536105745985515605895895255575435655535455415414994724594764894504304204364344024214053933903803443463613573543393103293513333473383323183563653613263053192963153403173703523163362992892952782552622922622702602832652512252652712772402502102381912162142382362242031962152312052052072211981992011962021992181851911892071611981672041591821771711822451992012102131661681872101591761991861821811811761461751711811601711831691791751561441741531481401451701751691931711631781561861641201611641521561681441401781371701411391501501621501451401551571581531321521741481481411431561531571431531571701621661281641361521251231281351171371251361131251251401381331231341131301401181171231261191151311081181251301571631211131251291291031261461121291321161251291181121311111081339812210411611612212792117115126139126117107119111138126118115118111100115971031031141079910193959210411510310410311612410813312311513010612512313410910111012497105106121116132104127111113879410710999119119117106120107104979712612812910711111511410010412113211210710212199119102768310810190878181759283908696911001009610791959610577101100988592867590988568928793909484968289808099918975828396906490777799528780698468859574759479103878396826978818191848689939181797988918783818878856568917264697062627764688180796483977680757968959261697678737852765871605670536564737074605564615570636065786557607477685851615856595546496167486460495663585565557087667667696971747184575068586461706056606060545654586170795661726062766477796582616565676957638066658358607269655455646127 363100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

935 855000000034 233 136000329 058 396000000000194 323 4410000223 057 9260000400 283 7890000767 487 9100003 012 147 64900510152025303540Phred quality score0G0.2G0.4G0.6G0.8G1G1.2G1.4G1.6G1.8G2G2.2G2.4G2.6G2.8G3G# 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).

99.8 %32 804 69599.8 %0.2 %

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.

99.7 %32 761 91499.7 %0.3 %

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%).

0.1 %42 7810.1 %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 %16 428 90150 %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.

98.7 %32 425 13298.7 %1.3 %

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.

8 %2 634 3638 %92 %

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.

1 196 17129 54617 86935 08625 69026 54530 59041 73016 58631 68013 51012 15116 59119 8269 57523 79515 75417 86723 82232 33332 86532 65937 38030 13748 79584 7384 854153 9767 4387 44815 86215 5766 42019 9157 7887 73013 20617 5844 41126 436434 31917 81617 46228 60025 05246 53038 27568 85686 34811 59114 53713 82116 3898 93114 53816 81312 63941 66313 49825 87029 796 938051015202530354045505560Phred quality score2M4M6M8M10M12M14M16M18M20M22M24M26M28M# 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.

99.87%99.86%99.87%99.88%99.87%99.87%99.87%99.87%99.86%99.86%99.87%99.87%99.88%99.86%99.86%99.87%99.85%99.86%99.86%99.85%99.87%99.86%99.91%99.79%0.13%0.14%0.13%0.12%0.13%0.13%0.13%0.13%0.14%0.14%0.13%0.13%0.12%0.14%0.14%0.13%0.15%0.14%0.14%0.15%0.13%0.14%0.09%0.21%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped