European Genome-Phenome Archive

File Quality

File InformationEGAF00001561661

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.

179 796 19155 151 00321 957 73211 375 9236 966 3274 822 3613 654 9502 930 7502 453 5682 114 9151 872 4951 673 5251 526 5621 388 9481 277 4201 182 1961 103 5531 022 577954 573901 354848 981796 101753 549715 808678 470644 055616 335590 333560 360534 828514 235491 444472 461453 862437 037417 174403 091389 273375 950358 693345 949332 973321 946310 588300 130290 919280 795269 664261 856252 101245 783237 851231 651223 216217 854212 049204 349198 264191 943185 846180 892176 628171 698166 266160 804156 937150 605147 474142 133139 826137 227132 656129 693124 996121 817119 633117 723113 580111 557108 439106 786104 644101 94498 21696 65994 09192 73089 84187 68485 01983 42682 09680 58078 38177 24575 24273 24472 76270 18268 79167 09066 18964 23463 08961 60959 84058 62657 91456 25655 87154 82553 74552 88951 19350 35349 14248 87448 06146 73246 03245 36944 63543 62042 22741 53840 99540 35539 01139 32539 09037 99437 39837 00636 28835 29034 12733 94333 43532 76231 99731 86131 52430 81530 04329 98429 31028 18628 41027 83928 00626 76726 42925 96925 69125 27125 39024 94424 63724 47724 20923 75823 49122 81322 46322 56922 56021 45721 01020 74120 82120 19720 19419 70019 82719 75219 07419 14918 95118 50518 33217 40717 89117 50917 17416 89516 83116 67616 14716 20216 15515 84615 76415 40915 18215 13014 88114 56014 53814 18614 21913 93313 74214 28813 49013 65713 07513 43413 14013 20913 14513 11912 67912 37212 44312 39412 06712 00111 82311 43711 84011 45211 17011 09111 02811 09311 09410 90210 55110 42110 1859 98210 14410 18810 2659 9029 7039 5419 6569 3979 4769 2969 0228 8089 0428 8678 7558 4388 4588 4408 5088 4018 3548 1318 2488 0938 3378 0988 3027 9727 9137 8587 7887 6327 4967 5187 6067 3367 3927 4547 4127 3047 2907 1097 0986 8456 8376 9437 0036 9616 9476 9736 5926 6836 5686 4136 3756 6196 4976 5306 1036 2946 2226 0415 9926 2006 0895 9236 0255 9715 8035 8425 7865 8875 5025 5115 6165 5275 3975 3515 3355 2025 1485 2415 2995 0775 0165 2275 0345 0655 1704 8924 9755 1055 0264 8954 7944 8754 7694 7124 7054 5244 7794 5754 5894 4514 6584 4374 3654 4194 3094 2864 4954 2284 4524 2044 3674 1424 2184 0054 0924 0164 1003 9274 0363 9964 0433 9183 8233 7823 6933 8353 8213 6323 6193 5333 6403 5823 5473 5133 4933 4363 4503 4723 3243 4003 3333 2613 3983 3303 2073 2533 3953 1693 2233 2063 1343 2613 2923 2433 0163 1153 0523 0223 0463 0423 1193 1653 0122 9763 0093 0032 8172 9252 8022 7392 7392 7672 7402 6832 6302 7432 7352 6022 6362 7352 5602 5052 5222 6262 5532 5172 5382 6502 4242 5342 5092 4352 4742 3182 3552 4622 3942 3022 2722 3212 2572 3112 3692 2382 2182 3282 3092 3032 1042 1932 1892 1022 2672 2062 0332 1292 1192 0862 1741 9602 0411 9102 0772 0672 0582 0111 9221 9901 9411 9511 9941 9801 8881 9551 9021 9951 9081 9861 8561 8401 9021 6681 7481 8341 7081 7091 8091 7791 7381 8081 7801 7051 7481 7041 6941 6801 6781 6431 6531 6241 6731 6531 5281 6751 5841 5281 6241 6321 5031 4931 5591 6471 6351 6011 4621 5241 5411 6541 5341 5351 4371 4431 4561 5161 6221 4161 4351 5631 4491 3861 4971 3931 4841 4641 4381 3231 4421 4011 3041 4451 3771 2241 2911 3151 4421 2431 3231 3611 2861 2831 3411 2071 3311 3121 2841 3951 3021 3171 2611 2381 3051 2261 1841 2841 2741 2471 2601 2101 2291 2191 2131 1681 2331 1551 1351 1981 1591 0451 1271 0951 1771 1901 0831 1371 1821 0451 0611 1471 1871 1051 0871 0911 1061 1291 0711 0401 0611 0331 0611 0861 1201 0511 0531 0249851 0401 0001 0281 0541 0261 1461 0501 0931 0131 0849469479879399701 0299979079399871 0269791 039898904930885879922953903902898928793839794860865857833850873828819828841840827872840803770822810792769776811752766721789758750748732786708747746736735727692791669686689753712706728693681718709683790746681697744745702697684619666662683760693689734680669679679665620641595623649641663647700699659667655616690568571592635607640579592619604581596627588617627568603546567585599603552566535563549606567562570609538534560573545534532513547533505507520521456522512486542561504549556479509536474485481497498560464525533485542510490511463555494457485529480510482476497466456497458435444431475439422442438450423469425437391406408424434417469402451453447432436422394413447420450411434447389438428413380412389389376391396362374370420389387363356358380338358336386348366362351348341348363340334371354370352322321320331362345342329316334351347343359336415312309310299335358294322345331316298302356300339314343314324305311295279282270301295298289282306300287264287328291276268260284261292260295298290287322296315317310296287264285321323253298332310278300316279248291301282277256244246293261268280295257250272277301237268263255253229236251234240260217 954100200300400500600700800900>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.

550 7310000000000000169 815 270000000011 383 3480000149 556 93000000457 567 7510003 164 977 77000000510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G# 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 %52 622 40299.8 %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.

99.6 %52 529 94299.6 %0.4 %

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 %92 4600.2 %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 %26 359 01250 %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.

85.6 %45 121 49085.6 %14.4 %

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.

22.2 %11 724 32522.2 %77.8 %

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.

10 006 21788 84621 574162 04644 783255 15384 284111 17624 117198 23658 95651 686140 89083 69327 595144 87226 54038 38754 94376 27419 268140 49661 38485 907149 760433 59532 174603 90835 302118 39048 50862 77025 187152 80518 81737 71048 97685 42727 339208 8401 587 95662 64953 28882 425133 261104 06774 60077 89188 735182 773154 823207 536729 03337 734150 75387 02845 436285 285104 70324 07736 090 624051015202530354045505560Phred quality score5M10M15M20M25M30M35M# 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.

99.87%99.85%99.86%99.84%99.57%99.68%99.83%99.85%99.87%99.85%99.87%99.82%99.84%99.86%99.77%99.84%99.85%99.86%99.82%99.85%99.88%99.85%99.52%99.94%0.13%0.15%0.14%0.16%0.43%0.32%0.17%0.15%0.13%0.15%0.13%0.18%0.16%0.14%0.23%0.16%0.15%0.14%0.18%0.15%0.12%0.15%0.48%0.06%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped