European Genome-Phenome Archive

File Quality

File InformationEGAF00002391383

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.

37 841 58371 975 048114 480 625160 741 485204 392 669239 344 829261 402 242268 446 717261 222 848241 990 983214 589 371182 839 619150 437 487119 711 77192 580 61069 755 97151 326 76737 025 27826 290 89018 426 22312 826 4098 885 0926 176 7154 326 5963 073 1022 244 6991 685 0921 310 6031 049 780867 895733 252631 507554 437494 163443 251401 686365 262334 412310 388286 297265 241244 776228 315214 375201 328190 126178 701167 927157 298148 706141 002133 277127 696122 238116 388111 911106 647100 63696 98892 36388 39684 39781 24777 60174 04771 75469 50867 02663 88061 94259 27156 64353 96252 05149 63449 16847 60046 41845 61143 49642 63541 33240 20838 94937 59036 36834 91535 20033 61632 66431 48330 83330 20229 84528 97227 98527 24226 47125 87725 02824 60724 34024 37623 76923 12022 77721 95821 34221 17620 78020 42719 89519 58319 31418 65318 64018 42618 00017 59217 62017 09316 95016 62716 30215 92615 53815 35815 10114 90114 68514 43514 24914 25713 99214 23313 90013 80713 54913 34313 16812 58712 36912 15812 12211 82611 73711 61611 42211 43911 22411 29811 08710 89111 08610 74110 90110 50410 28410 0119 92510 18910 2449 7559 7419 7809 4219 5179 0939 2098 7008 5648 3698 3157 9948 2807 9638 1948 2187 8777 6837 8947 5497 7657 7967 8197 3667 1097 1717 3116 9866 7796 6116 8276 7216 5456 5476 4406 6156 5916 6146 3616 5356 1856 2316 1966 0246 0536 1025 9805 9945 7315 7836 0775 9795 8855 8795 8105 6675 6355 7615 8165 7465 5605 4495 5355 4455 3905 2645 5325 3515 2515 2355 1495 0265 0364 9505 0354 7624 9504 9194 6084 5254 5334 5874 5764 5274 5204 6324 4024 2734 1254 2934 2534 2274 2794 2524 2364 0624 0844 0703 9624 0244 0593 9524 0654 0214 0083 9843 8043 8623 7193 9673 7763 6813 7703 7433 9073 8223 7263 6993 6953 5923 5113 5813 4663 3333 4223 3693 4153 4493 5433 4913 3873 3573 2943 3033 2513 2333 3593 1173 2813 4153 2733 2363 3993 3073 2513 2453 1283 0123 1323 0143 0603 0233 1633 0992 9903 0903 0313 0202 9622 9612 9452 9242 8492 9102 9122 9002 8412 7982 8722 7462 8222 7502 8372 6672 6842 5482 6412 6252 7212 6162 5332 6372 6682 5792 5592 5532 5442 5682 5012 4352 4802 5762 5242 5172 4952 4712 3602 3872 3822 2522 2992 1892 1882 2002 2762 3182 2842 1742 2532 1752 2292 2382 1632 2332 2812 1802 1822 1492 0582 2102 1212 0902 0322 1332 1182 0892 0992 0852 1462 0002 0671 9941 9942 1142 0222 0382 0972 0001 9961 9501 8991 9191 9691 9361 8721 9221 8871 8901 8141 8341 8231 8721 7261 8031 8321 7681 8711 6841 7351 7091 7871 7281 7971 7511 7711 8021 7781 7351 7411 7351 8191 7741 7791 7001 6571 7161 7511 6531 7261 5951 6161 5941 5861 6661 6191 6361 6101 5981 5601 6011 5981 5681 5711 6081 5601 5261 4871 5171 4851 4721 5231 5061 3691 4821 4501 5191 4141 4631 4871 4541 4061 4371 4521 3921 5461 4001 3191 4281 3661 3821 3701 3001 3841 3741 4421 4041 3851 4461 3951 4531 4511 3751 3811 3551 4991 3631 3731 3431 3651 3231 4351 3771 2901 3431 2651 2681 3531 2711 2711 3251 2461 2351 1691 2131 2341 2041 2361 2601 1751 1641 2041 1461 1841 1561 1921 1621 1341 1001 1711 1761 1071 1771 1331 1521 1011 0981 0941 1231 1491 1111 0801 0761 1091 1091 1081 0891 0841 0711 0741 0621 0681 0961 1231 1191 0771 0181 0481 0281 0611 1049851 0149681 0231 0199869921 0151 0269731 000937993949953974880999915914939968910887901882905863933871895834935913923880884902852880909907897881891917848922849832864901828869884793842878907842829806819822789913904872906879920860879913897873873813833809838819793787795766769800756797821806751775738752777759764742720772734750757707709760753765784735765786741770770778718693714695750710733668695717685702739694713694703696718755695694637659726665710687719699705688683685721680662718682697682638642710650690601663598599607668594623602596656624610580612698575609561545659609678600621601649613639652633667652573617569608603618607592573584566564586569538563528577527551604550540575585555576595576522553524551574512530533529519541522516574507551539531574533507574564532547556537522538491545519526579541561534503549572515583515542547532499515564513520539512588547586565538521465511520556505524523522498480490484527533530524566546495524502548494533521467482471511492517533516514490499491494494486516494475524553467470508550536467495497517507453535483502493463496521470466464459460447462452433417441471457448489453449415462465460478499491492473448475412424439427473449419456446440435422468459465438458458456449419401464463432434434439430410455415449413442429477399410502413419410390399407397413366390423387401480464415422382403418350401424 299100200300400500600700800900>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.

3 584 7430000000203 538 5010001 614 263 238000000000990 500 30100001 202 578 53700002 118 095 97900004 268 128 01300018 350 889 69600510152025303540Phred 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 %189 669 67899.6 %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 %189 135 30499.3 %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 %534 3740.3 %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 %95 203 90450 %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.

95.6 %182 053 53895.6 %4.4 %

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.

6 %11 353 0936 %94 %

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.

11 310 944233 548157 835267 533210 703215 810242 378312 863156 978241 273119 795103 542140 331150 94688 241176 547139 837156 042196 581268 222273 939262 325329 426243 297377 410572 09554 010946 98574 15868 577121 166131 93970 028153 42668 40067 498102 914128 36745 241183 5772 518 803142 874146 624229 723197 082345 461290 958458 024635 648114 939130 101113 246137 73780 282127 664126 59894 919277 378100 331179 473166 996 116051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M# 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.

99.71%99.7%99.71%99.72%99.71%99.72%99.71%99.71%99.71%99.7%99.7%99.71%99.71%99.71%99.71%99.72%99.71%99.71%99.72%99.7%99.71%99.71%99.81%99.43%0.29%0.3%0.29%0.28%0.29%0.28%0.29%0.29%0.29%0.3%0.3%0.29%0.29%0.29%0.29%0.28%0.29%0.29%0.28%0.3%0.29%0.29%0.19%0.57%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped