European Genome-Phenome Archive

File Quality

File InformationEGAF00004839042

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.

42 915 65683 853 206135 897 392192 587 291244 457 319282 301 742300 008 446296 640 218275 046 118240 995 633201 047 390160 338 085123 074 56391 160 71965 461 19045 714 24631 211 81320 898 81813 771 1198 978 2385 837 5763 806 6882 519 8221 691 8001 175 660861 501651 713512 603419 016356 146310 620274 309248 843225 435206 513191 029177 331162 905152 472142 562134 015126 715117 620111 030104 74798 62992 79788 26283 84078 89874 32570 78365 83461 96758 73055 68251 90748 27345 87543 07141 31239 02636 50935 26433 61531 93330 36128 85527 79426 28024 82123 91023 00622 40021 45921 09520 44119 77919 00618 61517 93117 52317 24416 76116 29615 98715 45615 37514 95815 01314 16214 01413 72613 63113 17613 41413 12512 39311 78311 67511 11411 18610 73110 66410 36210 2169 9099 8769 4959 2829 4209 2548 9779 0508 8028 2858 6448 4708 1187 9467 8557 7047 8837 7877 4567 6387 4617 2347 2617 0007 0946 8686 5976 6056 2456 4866 2316 1655 9295 8845 8235 7475 6305 5075 3755 4615 2355 2765 2585 3405 1655 2214 8884 8344 8224 6794 7444 6524 6104 5564 4324 3354 5404 3494 1924 2724 1553 9103 9703 8553 9503 8913 7383 8813 7713 7183 5433 6453 4893 3793 3333 3343 2593 2643 1503 2403 1833 1523 1573 0183 0862 9472 9643 0803 1513 1462 8333 0202 8892 8902 8132 7102 7282 6822 6932 6912 6602 6312 6102 6242 6482 5872 5282 4982 4432 4742 4542 4502 4062 5952 6082 4302 3612 3382 3052 3482 3202 2052 3222 3522 2642 2532 2962 2932 2642 2402 2582 2412 1232 1842 1232 0952 1682 1592 1342 2242 1602 0852 1472 1532 0122 1222 0731 9971 9801 9882 0082 0261 9091 9541 7821 9541 8221 9321 9331 9621 9081 7981 7161 7071 7501 7391 6141 5971 5701 4991 6231 6351 6011 6581 6821 6031 5981 5991 4971 4891 4461 4841 4401 5401 5331 4181 4901 5221 5491 4491 5291 5611 4521 4701 4241 4391 4651 4371 4781 4701 3651 3721 4511 3331 2781 3241 3561 3431 3571 3891 3211 3461 3061 3281 2941 2511 2151 2381 3711 2561 3201 2521 2591 2551 2551 2451 2221 1661 2561 2301 2201 2091 1831 2161 2451 1851 1221 0781 0801 0701 1611 0881 0611 0471 1001 0681 1021 0771 1091 0841 0681 1001 1171 1001 0661 0351 0371 0241 0661 1421 0601 0161 0099829669961 0121 0031 0599899391 0561 0501 0399609689759729661 010987955995916947889905914922921966910899918968916863921843888807887858841819902893820926849890856904894915868896895875902917859839860874886838795837834849818851819830828836867867947859816836811835751763829782780861756794739727778763758775734750717830766779765825725747714730675671690737666681695718686723695690700688649665658629610674661624627632664670625611670631620688620631574623640598569649572551583574561572614536588535547543527541573565576519566541530516500518487596554528505507524538508498508477488498495502477467484457500563463520511507478490526465501508541492458492492484449496456468452466520534457455459514480518443438435480443520425481484467477492426471471471477466490445482443442516496487509508487545510500476501497522451435486467450481473428463483456489452425461405410445417425427462424432435427471427453415417434370381377349373392412365364361363400386356377346347378400374368324322324372343389314380370334326311345354347324347324438348321396335330348346355298312327312325334322367357325353341333326348350339345315323355327314299308301323289316295313327320281304280280301292305301274296283267294282268291293309264282279300290295297293285274311295234302282252241272251262306332291261266250319279288277254269234255280275231262284314258256247278254233273263259270249241254257246277238246279235259241237256250256267260235237226229254252241245244231216215220220211210229220270231228236272227199224220240276224235215214216248239227217231211210216229182207226234224235242222205214213220198205227207218233212206216236246240222212213217233227216214233231254219205216207222218228231223192230255198204233222225220227204194226210213217200206219220220236185243208230206224195200217230202211198192207188199202214220218204232243252249231208205216217211210195215206235211230220222197229215212204189223198216207213225232189215220211215213247206214269 824100200300400500600700800900>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.

3 582 824000000019 482 3730001 028 164 811000000000593 725 1660000659 961 00700001 455 836 28400003 058 390 23900018 543 729 94000510152025303540Phred quality score0G2G4G6G8G10G12G14G16G18G# 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).

99.6 %167 220 96699.6 %0.4 %

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.

99.3 %166 784 83299.3 %0.7 %

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%).

0.3 %436 1340.3 %99.7 %

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 %83 983 02250 %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 %164 765 45298.1 %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.

14.1 %23 627 62014.1 %85.9 %

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.

6 744 091123 43673 221153 003104 929114 818137 162178 41382 920147 26560 20853 18582 45388 84845 058113 64574 03984 997111 290148 618149 756155 189188 234146 717244 917419 80124 123782 82635 38133 93975 19573 33833 87194 28235 02535 78463 63781 02621 357128 4881 903 92990 48086 624146 122122 287229 888207 619293 693537 15056 57277 60466 88890 21943 15381 94278 91257 339216 41858 622119 359152 892 175051015202530354045505560Phred quality score20M40M60M80M100M120M140M# 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.

99.73%99.72%99.74%99.74%99.73%99.74%99.74%99.74%99.73%99.73%99.73%99.74%99.74%99.74%99.73%99.74%99.74%99.74%99.75%99.72%99.74%99.74%99.81%99.73%0.27%0.28%0.26%0.26%0.27%0.26%0.26%0.26%0.27%0.27%0.27%0.26%0.26%0.26%0.27%0.26%0.26%0.26%0.25%0.28%0.26%0.26%0.19%0.27%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped