European Genome-Phenome Archive

File Quality

File InformationEGAF00002869129

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.

64 804 277132 325 800211 870 644284 614 657333 619 494349 711 015334 573 236295 423 517243 890 276189 744 975140 249 65099 043 17267 272 33944 207 47128 255 55617 698 24410 988 6796 785 4324 237 2272 701 6971 799 9531 246 753919 691712 182576 789488 334418 088362 006323 145289 445260 804236 509213 170192 414174 670157 421142 734129 928119 381110 910101 18492 08085 77779 59074 28169 26165 11360 55957 55254 95552 05049 09347 16145 75943 75442 05940 27939 12637 35935 66834 31033 30932 84730 92330 72729 22628 75027 15426 86926 20325 18023 79123 51722 93521 98021 56521 18620 34919 84819 32418 79318 21517 26417 01716 88316 21815 72115 77915 33714 69114 98314 55414 26714 09013 59413 71613 06312 52412 40212 14111 35811 42511 16810 80910 96711 15910 69010 28310 14210 15610 0189 8019 2459 1779 4049 0669 3939 5049 0358 4648 6938 2958 0358 1958 1408 0477 7667 5867 4357 8687 4577 3447 4617 2547 1136 9637 1947 1406 8636 8176 6976 4876 3616 4136 0175 9706 0005 9585 6255 7505 6635 8525 5805 6355 7745 5185 4305 5315 5245 4575 2545 1945 1354 9954 9894 8344 9304 8644 8764 8944 8774 6004 8134 8614 5924 6724 3284 3764 2834 2794 2434 2194 1273 9154 0784 0093 8513 8453 7493 9173 7743 7463 7293 6423 6943 6723 5953 6483 6893 5023 4993 5433 5013 5083 4133 3983 3143 2933 3643 3133 2363 1333 2573 1083 1993 1873 0962 9913 0273 0892 9232 9092 9112 8212 7792 6412 6852 7602 6632 6232 5632 6232 5982 5912 5782 6092 5162 5052 6182 4702 4542 6112 4072 4532 4832 4262 4602 4072 4272 4172 3452 2372 1032 2992 2522 2642 3172 2912 2942 2072 1502 1162 1832 1792 0002 0772 0271 9082 0181 9462 0072 0142 1312 0081 9341 9431 8651 8871 8571 8511 7981 8201 7751 8561 8261 7681 7501 7671 8061 7481 7591 7121 7681 7691 7001 7051 7131 7211 6901 5881 6561 7321 6681 6631 5901 6751 6441 6691 5771 5921 5861 5941 5681 4861 4921 6041 4991 5311 4431 4231 5051 4181 3961 5271 4791 3871 5291 3711 5151 3921 2971 2731 3041 3121 2991 3781 3421 2381 2641 3321 2091 3321 3461 2951 3001 2511 2591 2501 2391 2181 2311 2021 2471 1571 2371 2161 1851 1831 2351 2461 1741 2931 2231 1511 1621 1241 1121 1401 1671 2011 1491 1991 1781 1981 1941 1951 1621 1971 1751 0721 1051 1521 1231 0671 0661 1321 0961 0931 1061 0811 0921 0961 1141 0681 0831 0431 0781 0479899081 0081 0611 0081 0351 0029709719971 0859731 0029561 1411 007929931936948930923971917901921840881892910898877827870912851847832888878870906861914886878830858809890916788813821785817732769737788771754732741777819717858745765775787796751727800799816749741742755765757781738700763765778730702742725733708736680695672721696677721693780716711643684687644681660625680692791660646685626636648589661602617633601626641607594617648628606654643625603587587575594624600565615612541583591558611582591548588549559536551557537575549508590553549578508527522507561557542591530547525545585551512511479533517536471512509498481469492466487509489501488650533520498471487494512498493455496505527500522503493523498474492511517462489462463436470464425467465483480431461437422471439432480529506426457441459447439452445435410430455447396437455458402421478418427443465448417473459457457457451436389372406392367364373399369380376385367366363358374393379356385353414366397362373366367384388426411415437404400364373388424360412405371384363366357348404376350335337337362340409338357351324323319336378347358336331335361341386331349280327315329326313310346314304302317296332305281291323342321342343315343316312308300305338324315305319354323298328289319292306308318311325300290310286317326320339309305285307287316301297309327332288321301364327339304309326318296320301312274311341310300272270302262269275297280278273299271276262277307277287281285287301290267284278263261286273263264264274260265263261289297292266222242248275271238237226236254270279263268280265266268244258254289245228244221270272228224279245232244255229222249234245238258261256240237262267266227249239251234231240240260233253243240233222229245263269257251266230242265267235266248234270222248251236257276260250226257218216241252236233222218243249233249252241231252263222235224227236210239284 136100200300400500600700800900>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.

2 812 867000000023 098 2340001 094 458 559000000000621 626 2850000666 693 37000001 424 503 49000003 045 252 93600014 665 315 87700510152025303540Phred quality score0G2G4G6G8G10G12G14G# 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).

99.3 %141 660 02099.3 %0.7 %

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.

99.1 %141 416 59499.1 %0.9 %

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 %243 4260.2 %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 %71 336 95950 %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.

97.7 %139 384 84897.7 %2.3 %

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.

9.2 %13 117 8919.2 %90.8 %

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.

7 096 156129 77975 757154 374111 234118 912134 079186 89280 669141 11262 28255 71479 73992 00546 169110 73477 60588 632114 415157 589157 498158 172187 705156 522258 737431 10223 614742 76735 39634 49469 30874 88733 43991 95735 91937 22460 52080 90621 169122 2011 789 77284 21383 444137 662117 732223 664183 856323 045441 76956 48570 31166 47979 52541 87677 95276 89555 594198 17959 360115 313127 576 598051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M# 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.

99.82%99.81%99.82%99.82%99.82%99.82%99.83%99.82%99.82%99.82%99.82%99.83%99.82%99.83%99.82%99.84%99.83%99.82%99.84%99.82%99.83%99.82%99.88%99.84%0.18%0.19%0.18%0.18%0.18%0.18%0.17%0.18%0.18%0.18%0.18%0.17%0.18%0.17%0.18%0.16%0.17%0.18%0.16%0.18%0.17%0.18%0.12%0.16%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped