European Genome-Phenome Archive

File Quality

File InformationEGAF00001307700

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.

155 827 940288 765 056401 198 482453 133 727435 111 010365 670 460275 128 903188 527 178119 409 35470 828 38739 828 28621 533 58711 399 9446 055 1083 321 1811 931 5581 221 108858 570651 377517 872428 294363 577314 650274 071239 131211 670187 671168 296148 172132 805119 933107 43097 73288 52380 87773 89167 59961 80256 96152 63148 93145 05842 84339 85938 57235 74234 06632 00831 10329 96927 77127 06226 37924 81424 24522 91321 09220 41919 50019 06718 61017 53916 84216 20715 56615 34314 89013 82413 91613 88013 52612 82712 58211 90911 55210 94210 90610 81210 64310 0249 5559 2359 2389 0038 8118 5437 9937 9077 8777 7977 6857 5527 4377 1216 9626 7546 5796 5946 7366 4046 2566 4316 2265 8855 8535 7005 6805 3895 3164 9805 0234 8704 7644 6904 4254 5504 3944 5654 3074 4264 5614 3584 2004 0164 0243 8583 7183 7183 7923 5713 5363 5863 3063 3973 3093 4333 0893 1673 0683 1073 1372 9903 0003 1392 9872 9782 9452 6942 9072 7462 8142 9552 8932 8392 7382 7002 6132 6602 5212 5882 3622 5232 6042 5722 4892 3892 3372 3152 1882 1342 2052 0922 1622 2462 1572 0782 0932 0982 0672 2032 1222 1662 1332 0561 9812 0032 0121 9672 0501 9841 8941 9321 9051 9411 8781 8651 8811 9611 8221 8441 7791 8721 8711 8591 7211 8031 6781 7431 7451 7091 7791 6691 6131 7241 6181 6601 6271 5231 5371 5381 5431 6031 5271 4441 5161 4781 4251 4781 3741 3641 3801 5031 3681 3171 2871 3721 3031 3791 3841 3241 3341 2311 2221 3141 2631 2621 2701 2581 3061 2941 3671 3331 1891 1961 2181 2251 3401 2291 2171 3281 2641 2271 1061 1201 1761 1141 1601 1241 1531 1121 0491 0821 0511 0891 0481 0311 0119711 029924979884884945904938955919921841947929847929935856845962856790862843808844827868823869869844788803794797842757761806820853760783765835771827792797791813768764853802753789803780801809781798841735807804713736770762746742666740736795760674684696701716702711718712708780699736703737765689665643640599602649631644608690652651648664668630638645597644621608598608595597606566575580592567574526570526485544521536548556520508518517488483497504498523497489459479498530517504535502502502504467489482483446480445435473448430445509417439460450449430445418421406457428440459501475445454423429469490486435458460472416448483410443434434424425435447450395351410427416395422427433469488411436403426452410425382451387408421449397400366423378414358435448423395458439420425447379389391390405391378361388376343341364367334337369387393402400384380396410382365391359403350382380353349361363344365339308302328322329326306307335311304331366299315322321281296358286347343280325348355371360331338299297332313306335347305331339287311336339363323284339332339326316355307303288247267289280274312331330289319319294300287274277311265294289269282293253307307297271308268275265269268271253259261274240239240268245269273250266267245244284260277269248271255263266233250256252262250242250248281268259263270268275266255285235264276272276272252284253284253214228289222227240244251225235214219232229203238248239235235207195196214222223211215200199187206199198228195194218203216218230213228200190192212223208171215182169178198172205195172187208221169194173217193180161192183185172192202189208187205172182193201187179214188179201190209183173180184179192182195177169161191193200184177174186207179196185197217184202171187171184218194207163189186180199188220181201179179207200180198216227175210187186180197161173180188181192193150168185175171176138176155158157181167170177175158203188165175151197175155182181168176148154171177165176170182159165172165206176173189156184193157182159192169180191168158175189159179189214202206199200182179198185170196192192230177207196197190180220186187166188185178186178184172170172191182178182187170199178182194191169185195183147168191185169212197193183191216220221193174207211120 501100200300400500600700800900>1000Coverage value1k10k100k1M10M100M# 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.

1 132 78900000051 422 221000630 021 6300000000000331 299 8480000379 003 1320000996 775 47000001 841 274 522000010 663 239 26800510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G# 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).

99.9 %98 563 16199.9 %0.1 %

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.

99.9 %98 504 15899.9 %0.1 %

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 %59 0030.1 %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 %49 318 44050 %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.

97.8 %96 418 20097.8 %2.2 %

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 %8 103 3088.2 %91.8 %

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.

6 720 811183 38886 784247 892102 863107 556329 364146 22670 144134 39952 58045 226125 70562 39134 45279 00148 97554 01585 22781 90587 47493 396100 41978 885133 803211 22923 591421 80628 22527 15353 74045 98329 43955 21626 99826 54139 41850 29019 83775 705997 49455 07854 44484 72671 845127 440115 851165 333297 75034 98048 04542 34055 50329 09548 57548 64136 257124 92436 75071 69989 834 764051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M# 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.

99.94%99.94%99.95%99.95%99.95%99.95%99.94%99.95%99.94%99.94%99.94%99.94%99.95%99.94%99.94%99.93%99.93%99.94%99.91%99.93%99.94%99.92%99.95%99.98%0.06%0.06%0.05%0.05%0.05%0.05%0.06%0.05%0.06%0.06%0.06%0.06%0.05%0.06%0.06%0.07%0.07%0.06%0.09%0.07%0.06%0.08%0.05%0.02%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped