European Genome-Phenome Archive

File Quality

File InformationEGAF00002336701

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.

115 935 649211 125 378300 784 864360 666 551379 178 933358 274 337309 782 737248 319 420186 910 157133 106 63590 382 73459 055 53637 358 14223 021 90113 932 5868 364 6955 031 8683 088 0841 958 5321 298 357910 241675 534527 693432 060366 581314 972274 582246 474219 619197 614181 258165 593150 587139 064126 570117 072109 155100 30391 13383 47977 19072 46566 56961 62856 67554 16149 99446 67643 19940 14438 37936 64434 51632 47732 01630 26929 05927 48326 80524 23623 30123 06121 81821 46020 46320 37719 08118 86718 33417 38417 28216 86515 81414 92014 73314 39314 34313 83513 36713 43212 38512 20211 73011 76411 29211 28410 87510 32810 35810 0329 9709 8139 6229 3359 3328 8898 7508 5178 6828 3978 1178 3668 1597 7947 7947 3887 0987 5657 2187 1766 9526 9836 8187 0466 5376 4686 4426 4856 2356 4966 2936 2615 7795 4835 5605 6055 4715 5205 2095 3255 1854 9495 2455 0684 9144 7244 7204 6354 6244 4624 4344 4704 5834 4164 4444 3604 4294 3264 1454 0653 9524 0013 9403 8823 9073 8003 8893 9383 9493 8563 7143 6663 5453 6243 4953 6133 5963 4093 2003 1753 1383 1973 3703 2503 1923 1543 2233 2583 1613 0883 0822 9152 8372 8562 9342 7802 7582 8112 6702 8162 6922 7952 6142 6972 7382 9062 6112 5862 5162 6032 5772 4772 3212 2862 4452 3512 3372 4472 5152 3562 3362 3302 2962 2022 3502 1732 0702 1472 1352 0872 0552 1442 0022 0682 0472 0142 1481 9972 0331 9661 9782 0931 9671 9421 8861 9221 9151 9421 9592 0482 0561 9531 9221 9802 0031 8771 9971 8891 8501 9281 8392 0001 9011 8051 8961 7391 7641 6681 7191 7241 7441 7211 7881 8151 8051 6611 7281 8081 7471 7801 7341 7321 7111 7531 7771 6621 6431 6671 6941 7131 7431 5991 6131 6861 6971 6551 5661 5601 4701 5521 5431 5161 5781 4661 6301 6821 5191 4611 4291 3781 4301 3651 3341 3951 4021 3921 3241 3431 3281 3551 2691 3551 4051 4081 4261 4191 4441 3261 3691 3681 3051 2421 3101 2571 3171 3291 2891 2341 2521 2371 2011 2171 1731 2411 2091 2801 1661 1611 2781 1981 2351 2221 2031 1751 1211 2201 1101 1211 2211 2471 2521 1691 1661 1351 1321 0971 1121 1371 1161 1101 1161 0711 0751 0631 0461 0631 0071 0251 1061 0131 0751 0671 0051 1021 0811 1541 0751 0981 1211 0721 0351 0251 0351 0601 0621 0281 0281 0201 0449829469629639849549791 042994893958970884989901938896866869834980915851849882844854897837817821934980902917851849886856835833868793756791788771800719777779791799736808746790817783812750766811784779762797717757697708728639716703749741726701702680682665679767717710644650666616684616668643628655609663624625595617610648612637616651571612606618554657623602659590553616624577544559562577584559582594569548530557531516523557559527591554520537499602536474519472504513550537500552522501509530534529490482474500508489492490493469436481487461477497484492480478510445447461447455438446440449416459414431424409421433405442414425437454434414438407427400420425408432441444453449443377412381411414423408424413383434411430436413391384421395345448412396374411421417430429412436465438395402410395403363377397405389404427372400414416386377377416398382370393393396361415362397402414362372350388362358348373346383326370334346363365336351379360349395359308362386412303359355359370374400344341345365333313341358342363502367337310325311342309347397338336315373335334349349362316346332318341303317312307322337322320315329354322343328339326307295302278304269271259288252310275279284245255271259303296272258243278271318275271273274275286283273251240259239233233243283255245231240259247237255229240246260257251242232224218216236254257254265262267209235245233190252231237242263225224263240235220246225247239233244231210224232234239204236273274252250265239217233243238231231219243193234220227204247227257229216220240227209226239208202207235191229206222221216197195196201200237216242201216231213233212231228239214215216193217188195197201212205161184196200188204234203186214197205204178204240190172182186195202194237214202203195225215196187200203209199203237216196155186187198179175219188185179194190190220176170201162176181176163146184173197196174156186153172188179226 730100200300400500600700800900>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.

2 705 070000000035 829 478000823 120 920000000000460 057 0650000513 777 27000001 080 882 95600002 249 005 20500011 612 530 11000510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G# 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).

97.3 %108 142 96497.3 %2.7 %

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.

97 %107 761 01297 %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%).

0.4 %381 9520.4 %99.6 %

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 %55 555 98750 %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.

91.7 %101 937 58491.7 %8.3 %

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.

6.6 %7 349 3616.6 %93.4 %

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.

10 337 677153 171121 917190 756164 578181 819205 945259 137198 566226 019130 882102 936108 561101 99966 447109 87481 64893 563112 567151 273153 372181 110238 409156 324227 069314 36176 484508 09982 98467 79699 371111 492108 361126 84073 33277 43687 847107 59658 761161 3321 230 362102 689106 349162 422116 013180 898184 137220 409381 07387 80793 63989 499109 45769 610203 24992 95983 649162 93589 897132 626104 415 092051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M# 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.

99.68%99.66%99.67%99.68%99.67%99.68%99.68%99.67%99.67%99.67%99.67%99.68%99.67%99.68%99.67%99.7%99.69%99.67%99.72%99.67%99.67%99.67%99.74%99.56%0.32%0.34%0.33%0.32%0.33%0.32%0.32%0.33%0.33%0.33%0.33%0.32%0.33%0.32%0.33%0.3%0.31%0.33%0.28%0.33%0.33%0.33%0.26%0.44%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped