European Genome-Phenome Archive

File Quality

File InformationEGAF00002797083

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.

14 352 09731 760 10459 430 45198 506 920147 812 790202 761 397254 508 416292 743 954310 159 984304 179 977277 602 958237 446 287191 187 845145 795 572105 754 64873 420 41548 910 87131 562 71919 815 71812 228 3457 500 9654 652 9032 969 4711 985 2111 410 5691 074 065871 463733 193640 367561 277498 075447 429405 488367 191333 948306 129281 963262 094244 207224 265210 370195 509185 636174 483166 660158 590150 063141 360135 769129 390123 028116 843112 155105 982101 03895 26291 48687 40083 19779 65473 94370 07166 70963 97561 20858 59355 28852 78550 31947 99845 70743 68841 85840 17639 42537 75136 28734 92634 23031 98131 04230 77929 70928 94028 31927 78427 11026 34826 49425 69524 74824 29623 69723 12822 39622 38522 03821 31121 11320 47419 80819 50319 05718 67818 37417 76717 33517 14116 48116 08215 70015 39415 19815 06415 32314 81114 37814 02014 00113 45613 05912 84812 99312 64412 46512 30812 12612 03211 68411 48311 58311 47911 45410 99310 89910 89710 79910 43110 25210 05110 13610 21210 0329 8349 5399 4609 4239 1399 3119 1619 0319 0598 8248 7268 5748 6718 4058 2248 2138 0738 1747 9528 0737 9267 6867 6187 3057 3397 1957 1107 3057 2896 9937 0026 9416 7746 6296 6366 5056 5306 5636 4616 3416 3016 3896 4336 2965 9785 8836 0055 9425 8865 8415 7625 5175 4775 5775 7005 6045 3815 4345 3795 2475 3145 0495 1975 0664 9034 7304 9014 8834 6304 4674 6974 5694 5854 5874 5534 3854 4874 4704 2354 2924 4384 2634 3334 1564 0824 2474 1594 1604 2084 2484 2674 0463 9264 0193 8903 9013 7683 7533 7493 6963 6343 6833 7503 7263 7243 6823 5723 5533 4923 5193 4613 3943 3673 3153 3003 3003 1823 3123 3003 2833 3613 2313 2673 0893 2213 1743 1643 1683 1813 1523 1093 0273 0143 0752 9082 9152 9382 9192 8542 8642 8932 9783 0353 0083 0123 0242 9922 9352 8793 0362 9542 8172 9052 7942 7372 6832 6072 5342 5152 5162 4972 6002 5412 5882 4662 3382 3862 4772 4272 4352 4292 5142 4082 4192 3322 3632 2822 2392 2232 1382 2352 1412 1062 0442 1192 0692 0522 0422 0561 9642 0042 1122 0912 0212 0312 0411 9771 9552 0002 0061 9511 9821 8101 9731 9981 9171 9961 9151 8601 9381 8381 9121 8971 9041 9311 9011 8131 8041 8291 8801 8481 8381 8191 8661 8611 8111 7701 7601 7051 6831 7331 7481 7121 6591 6541 7211 6101 7201 6801 6661 7371 6381 7151 6101 6851 7341 6861 6791 6631 6661 6051 6471 6411 6541 6111 6011 6441 5171 6081 5451 5681 5471 4791 4611 4971 4491 4071 4091 4381 4171 4541 4311 4691 4381 4721 4931 4601 5111 4611 4141 4291 3301 3701 3561 3631 3671 3861 4401 4321 4181 3661 3731 3011 3541 3511 3611 3051 3661 3641 3211 2441 3071 2171 2331 2361 2581 1761 1961 2661 2281 2131 2361 1601 1551 2751 2561 1891 2031 2111 2001 1431 2161 1731 1561 2091 1481 1571 1941 1851 0931 1041 1281 1241 1181 1311 1241 1471 1491 1001 1661 1301 1851 1431 1441 0771 1421 0901 0881 0921 0841 0921 1021 0609469921 0439901 0049799449371 0001 0318969819589229601 0121 0209971 0409839619879579479979849741 0069899529589721 0011 0139699429659681 0019609591 0259428859501 020999963901916963972921843891890886908903903898914917849875833852894885821838839843830814888815839788856802826743890805752817784786773779755762843818816756725773755806771807699738755752781743727685642768749742694750754749740722657717727672636746678672674693714739758719652664669648687696732667655647636619612592585594583608609554570572576622637591603641609652598617575554566587592532634552551590585592535540544534513495522550515495512487508510541578527474557504534498485492504523497478513485481460472453486478463507476473498471476468497474531563510536510444471477478469415443452509440475449443450439442470448455503468427441405471433456454433449441477469463471421456431466396438417451438379421449440458452427422401419444407443425420426393394428403446403427414434425405423436409378411427397448413427415435392420416402416387420391362363417387401421387387399407396392422398387405379392365398374372389374369342398375390327383378352356351344370347375332396367359360356379400383379361393400392359388388334359377368414368393409362406370374369375347401358385367402362382389370359371365379390374365369370353348359349350347343291324359356335362323374373324338301363331340319335339355328332353342341352350315334356341348339355336323336322316331286352327350337317306301305332330306314354329304280290293305329320339337340336315312312327348363362372348319294298297318321313335312345285317304399 681100200300400500600700800900>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.

4 778 1270000000110 702 0230001 349 288 250000000000770 697 3850000853 194 80400001 833 816 46200003 760 989 17900021 761 769 54800510152025303540Phred quality score0G2G4G6G8G10G12G14G16G18G20G# 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 %201 219 49299.8 %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.

99.6 %200 841 46299.6 %0.4 %

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%).

0.2 %378 0300.2 %99.8 %

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 %100 812 03950 %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.

98.1 %197 727 62298.1 %1.9 %

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.

7.8 %15 744 5407.8 %92.2 %

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.

9 113 627216 755134 823250 869189 858195 683220 157295 224133 880225 461109 68592 592127 266144 36176 780174 257132 208143 873173 242229 287237 103237 859268 445202 508325 235549 91939 644997 69458 36956 617112 725112 30649 793138 00156 82654 44590 594122 59433 038188 2072 776 502121 227112 295189 137156 985295 371262 520383 723652 42576 30099 64890 845113 15054 61099 467102 62573 407279 66979 129155 140179 891 928051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M# 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.

99.8%99.8%99.8%99.81%99.81%99.8%99.81%99.81%99.8%99.8%99.8%99.81%99.8%99.81%99.8%99.82%99.81%99.8%99.82%99.8%99.8%99.8%99.89%99.76%0.2%0.2%0.2%0.19%0.19%0.2%0.19%0.19%0.2%0.2%0.2%0.19%0.2%0.19%0.2%0.18%0.19%0.2%0.18%0.2%0.2%0.2%0.11%0.24%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped