European Genome-Phenome Archive

File Quality

File InformationEGAF00008413786

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.

93 598 700158 550 723217 870 244261 903 587285 316 107288 436 433274 644 974248 919 410216 750 226182 301 038148 843 929118 498 05192 188 64870 363 26552 774 13839 024 67128 504 68320 581 94014 762 41210 509 8877 433 5585 264 0543 723 6162 654 0301 900 8241 385 2081 023 913780 266600 531468 465384 042319 608274 151236 549211 392190 465170 641156 117143 905134 801126 297118 814110 701103 57397 68290 54486 24681 29176 57971 68167 76263 89160 03756 78753 32250 42147 39345 70843 64241 10238 56537 31235 07134 33233 02432 20430 59330 20529 04527 53126 66825 86525 34324 69723 39422 20521 80621 31320 80620 32419 20018 51118 29617 86617 75316 90516 44216 44415 92715 44315 27515 12514 91014 49214 01814 04613 81013 43713 04812 76712 92312 27311 87611 65611 64111 35011 24710 58210 67210 1099 9819 9409 6819 6239 7349 1979 2838 9308 6208 4918 4068 1318 1658 0848 2217 6447 7287 3287 2657 3437 1177 0716 7146 5696 7536 7146 6076 4116 2106 1406 0846 2036 0776 0316 0245 7955 9165 6795 3965 4485 4365 4115 1424 9115 0194 7374 5484 5844 7144 5864 5854 4984 4544 3314 2074 1324 1264 3744 1754 1184 1633 9653 9224 1234 0223 9003 7503 7233 7983 9293 6933 7493 7663 5833 6563 5003 4503 4593 4023 3653 4543 2773 3353 3473 0143 0913 1512 9842 9583 0483 0312 9682 9313 0112 8992 7102 6882 6952 7162 7812 6202 6152 5982 5662 4632 6422 5002 5792 3812 5492 5222 4912 4112 3862 3512 3392 2132 2492 3232 2782 2042 2672 1772 1812 1912 1722 0492 2172 1622 0172 0831 9211 9701 9482 0841 9771 9131 9401 9181 8461 8061 8281 7931 8661 8381 8811 8561 9171 8831 7681 8331 7561 7431 6451 5541 6171 6551 7591 6041 7511 6651 6421 6311 5001 5181 6231 5671 6051 5881 5471 6281 5601 6381 5921 6001 6041 5521 5311 5111 4571 5051 5281 5381 5311 4781 3681 4401 4501 4151 4451 4961 4261 4781 4381 4851 5641 4711 4441 4461 4921 3701 3511 3691 4111 3191 3631 3851 3261 3321 3671 3271 3251 2271 2741 2291 2961 2661 2621 3181 2561 2681 2571 2101 2381 2841 2051 1621 1941 1951 1171 0921 0801 0941 0541 0831 0761 1471 1011 1241 0871 0611 0651 0261 0391 0301 0361 0239879689841 0189939869951 0329799159611 0339199749851 0181 0501 0069641 0439371 0019509289811 0591 0481 047998927917930968953892896953918903807900885878876797912957853894901869844873847821781821783824813826766828792807814803812775774795741755806735751741763759706738703732771681679682710694644744696678674677673615724594649613628636613601643603615589602600583612614633601553511577532563538578534526572572576534523589553577566612593591584540547599556568570569546578546600556558586571585561556541541517531533546546546512539547536533543575538518561544506562555575544537506526526515530520591521541484498547508504499487521465509466450457452485469476458454518470471467443447425412488460396469465474425421447464406447428445433396443455419464410418443434425442500422472471473474414437500423416435426396420433430427472464432437393404397417425425487441417401422384398392360387361395369387415398408406435395399384419444426375364375422377363401365388393385426402414386402376398394397402390387399404377464380398412368410396331360394410396400377472376379372455377411380366385363356391378364339343341352351363351337355387359316410397343354345346336336348324342354377375363383386362350358350326310380364356379378375370398332341331310321311299293329285286339322299301351297315301361345327303316330301291288292294263279296296293323306249281280286286235249292300231292258279280291272264254293299284258252276255266229245264254227237247269247261251266271234249237233252269219237243263227273256265285250238227231258236246238233238233241243206228189239223210219233241214233187216202213212227216268216204215233236219251230218247228236247237234235208219226202213208206218261202224227201219220239218218225180236206232223204209208221218216199196206222220203215203216193185191196215192193209229213212190202235229212205176220193204215218221218222199218221212218213232206225251251218213248200200177209238208194199217208200225215185220172204198189183222201219201197206216191184203184249 104100200300400500600700800900>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.

4 126 891000000051 024 496000681 975 995000000000420 602 2390000531 957 02600001 204 613 22500002 614 756 06700017 052 317 32900510152025303540Phred quality score0G2G4G6G8G10G12G14G16G# 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 %149 099 93199.8 %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 %148 973 76299.7 %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 %126 1690.1 %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 %74 706 53450 %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.

98.3 %146 942 62498.3 %1.7 %

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.

12.9 %19 257 81712.9 %87.1 %

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.

5 974 495110 94465 931133 65793 551100 487109 296156 31172 901125 82354 78148 49664 64577 37940 17698 47267 42077 48694 195129 761135 092137 676170 212134 271214 710366 68721 318681 20430 45729 99057 98564 64430 22981 22830 94131 61448 62467 76218 642110 0731 575 81076 44469 065126 923103 031199 059171 877278 612457 50847 41165 08955 87674 26633 57366 35664 02543 485180 48544 19099 493136 685 961051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.92%99.91%99.91%99.91%99.91%99.91%99.92%99.91%99.91%99.92%99.91%99.92%99.91%99.91%99.91%99.92%99.92%99.91%99.92%99.91%99.91%99.91%99.94%99.91%0.08%0.09%0.09%0.09%0.09%0.09%0.08%0.09%0.09%0.08%0.09%0.08%0.09%0.09%0.09%0.08%0.08%0.09%0.08%0.09%0.09%0.09%0.06%0.09%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped