European Genome-Phenome Archive

File Quality

File InformationEGAF00000584982

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.

51 167 65472 208 93883 195 81989 946 11598 665 018112 171 197129 929 250150 303 973170 868 656188 942 180201 868 116207 621 705205 203 384194 485 174177 054 668154 724 094130 097 809105 290 49982 257 84362 032 47345 350 66532 156 11922 209 31114 955 9189 857 8586 393 0544 118 4602 663 7971 746 2041 178 373835 660622 174489 524404 687349 288309 711282 798259 945238 519223 290209 369197 841185 790173 068162 851155 031145 958137 586130 407122 012116 036109 014102 27096 76890 60385 67579 90076 13271 95968 37165 21761 84058 58556 00752 28850 14448 23246 04243 58841 89440 63538 90137 11135 32734 26533 42832 39430 78629 99028 49327 81327 21725 80224 75624 18523 55522 31421 62721 03920 73919 94319 42318 53917 99617 12516 75216 08915 64915 10214 60314 26113 66413 55312 86212 52412 45111 99211 21010 98111 02010 70910 56010 1369 9219 5419 3109 3059 1278 7998 6008 4378 2498 2077 9447 7787 4677 2597 0346 9046 7136 7736 5846 3366 2866 0586 1105 9135 8415 6065 5185 2925 0525 1394 8914 6554 6964 6484 5084 5544 1594 1113 9423 8563 7643 7853 5793 4253 5583 4293 2813 2863 2793 1513 0712 9562 9862 8182 7562 7312 7312 6152 5652 5092 4332 4302 3942 4442 4092 2602 2822 0572 1482 1771 9811 9862 0181 8241 9541 9901 8441 8551 8351 8081 8621 8611 7481 7111 6781 5641 6161 6501 6441 6201 5771 5361 5161 5041 4621 5261 4911 4871 4961 4591 4741 3611 3751 3821 4711 4861 3121 2791 3401 2781 2891 2181 1971 1341 1631 1491 1261 1551 1491 2351 1431 1241 0651 0901 1491 1051 1491 0711 0651 0471 0301 0401 0321 0941 0391 0889901 0351 0149351 0459449679689459049149468818668809088388287867908318228097647807857537687667147527036997296386707307247157137186366246176456856206466655776216086315876065915745655275505635355415375025554974824674655155045284935094854614584954904544634614804535074714934744654584724114674734244334484844065124704964914354584304404294084084394254404384013883974143904013913953793694033893633493764083973714003923984283843713663953873403383563743623893673753023533703133083483233443103163423603233523573343293313393383613543333283062723112692592712622542432542732652962993063132672602472592532502652472352512482572612922412712952672672782892332202722472592742382342392262572362392112332272752462512082192242032212182211972232252082202391972272152032422062032061862012472402332172312182302472122191902081822182021991771771812082002462072252172121782331762081971801701821791821892011751892072071662002042281751891892302172051912141731911731781841701921661601501852092511671791781701621631651681451571951901801631771561721822281881841491471681461411471471251531481801851531581721641491531521411771771461841511481401481571701491601471321291681511481571621321681451401421231491521291401261561531341081391581701431731611681431411241631601571421381681671661351311431471451571571441521351491711971441351361491401351491331231591281331361301781261411381271291351441461251191221261171371051221211431471341421391421431531261481341371421221201041341221211509613012411112311012511711313810111410911411110011210910711310611712112710013111411010012113810310611213610611813110411112310211810998103991181061161097894111104941021059212497101948998961031041049310712013593113100122999895108951001211321071071221079195112959799101941031119899849590961099611611110213713511189901001028484921051089489104921128790909394100829010485106921019382908789878795931011058611082911129911010185969485961119286877410093981111161241041128882107958693858794801027589931028595789486871031091099110010698939487961017480818091899694928910891928787839710887109901011008290105123132989483849710010192848410285897998777084938989101987482967982728681778575156 951100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

7 094 2552 564 5553 836 3678 158 51512 708 0445 648 51191 880 17579 880 62520 328 5507 828 32512 049 11457 996 994198 928 011309 158 03671 179 19468 387 75658 902 31931 363 86624 881 21028 920 45629 184 56267 721 87779 603 808152 807 113509 168 366607 157 162570 839 973317 811 436392 659 782151 538 375371 460 937736 303 203943 553 1301 419 895 2961 573 561 5602 709 982 1655 008 931 3777 698 570 5388 118 072 0802 490 528 542730 516 864182 448 54624 766 03000510152025303540Phred quality score0G1G2G3G4G5G6G7G8G# 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).

95.3 %342 831 52195.3 %4.7 %

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.

94.5 %340 220 70894.5 %5.5 %

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%).

0.8 %2 610 8130.8 %99.2 %

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 %179 943 88850 %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.

93.7 %337 351 24893.7 %6.3 %

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.

8.7 %31 458 3518.7 %91.3 %

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.

27 629 494123 19645 513203 70359 80384 52962 19684 03871 017700 966286 955227 700541 174125 468128 6071 112 249155 9781 111 406365 35296 258596 88932 191242 7931 359 23717 552244 90518 01018 37920 71714 775 26526 94118 73019 95625 75620 01227 734781 5615 826 49965 08037 67695 32683 88042 682157 86059 48677 282429 072100 982111 138115 854184 37275 726209 274156 728227 508344 922680 884299 343 315051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M180M200M220M240M260M280M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped