European Genome-Phenome Archive

File Quality

File InformationEGAF00000827122

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.

93 597 21172 401 04634 435 42123 831 2148 615 6287 995 8182 835 7693 195 4241 352 7101 446 609760 649751 083490 302445 402336 467300 312245 799220 295189 947170 321150 333135 248123 336110 38199 81091 92484 72278 92371 82767 80462 55557 17753 61850 64547 84144 46641 90739 81037 33135 25733 38031 85130 04528 34227 06726 24725 23723 51222 61521 52820 72019 81919 13518 12517 53416 73716 02115 78515 20114 72214 49213 93313 38812 54012 34711 89411 61911 37510 77211 05210 46510 3489 6999 4819 2788 9088 9328 4988 6658 0588 1917 8217 5827 2647 1987 1647 1686 6996 9246 3736 4526 2136 1055 9396 0255 8455 5765 7265 6375 2735 2925 2915 2114 9614 9514 8254 7334 7194 6814 5584 5474 5234 4614 2504 2734 2804 2594 1804 0463 9193 9103 8353 8673 7583 6153 7533 5653 6333 4833 4973 3993 3723 4753 2853 4533 3153 3473 3173 3133 1813 1853 1643 2103 1063 0572 9012 8902 9482 8982 8532 8192 7022 7242 6872 6752 6572 7362 6032 6982 6802 6372 6632 5672 5312 5452 5022 4472 4052 5652 6492 4092 4852 3162 3572 2762 3232 3042 4122 2922 3602 2402 2802 3322 2552 2132 2022 2612 1162 1852 0852 1572 0672 1441 9822 1082 0942 0801 9602 0302 0151 9781 9531 9821 8651 9871 9511 9221 9671 9891 9421 9301 9101 9171 9021 9172 0141 8631 8591 9471 8111 8131 7951 8321 9001 8411 8221 7681 7501 7541 7101 7181 7331 8211 7761 6951 6681 7781 6521 7011 7511 7771 6581 6951 7221 6851 7191 6741 7021 6511 6651 6571 7051 7591 6571 6621 6581 6191 6491 6131 5811 5491 5561 6621 5891 5501 4701 5831 5791 5241 5631 5731 5031 5241 5801 5061 5141 5571 5221 4441 6231 5611 5311 4841 5051 5201 5131 4421 4691 4101 4061 4791 4981 4171 3741 4151 3911 4321 4651 4441 4201 3421 4301 4121 4431 4161 3541 3631 3751 3891 3381 3531 3981 3511 3831 3461 3561 3991 3661 3461 3801 3491 3251 3101 4161 3841 2891 2911 4021 3561 2531 3791 2841 3521 3301 2511 3021 3361 2941 3041 2801 2541 2831 2581 1851 2951 2591 2551 2501 2951 2581 2271 2561 3041 1731 2611 2091 2861 2541 2761 3021 1691 2201 2381 2161 2251 2461 2121 1651 1641 1931 1921 2071 2291 2441 2201 1791 2021 1541 1361 2241 1261 1481 1331 2411 1531 1251 2131 1751 1661 1391 2081 1801 1811 1711 1411 1241 1671 1371 1381 1091 1631 0921 0921 1061 1361 1221 1661 1181 1131 1631 1161 1061 1371 1291 1341 1561 1031 1631 0551 1181 1391 1271 1161 1411 0491 1111 0651 0991 0641 1081 1171 1051 1111 0861 1331 1131 1391 1191 0811 0511 0591 0811 0861 0631 0521 1411 1061 0801 1071 0381 0651 0531 0891 1011 1031 0551 0051 1441 0811 1301 0321 0841 0571 1181 1001 1081 0371 0411 0491 0921 0271 0621 0691 0471 0811 0531 0491 0401 0361 0651 0191 0331 0569889971 0451 0109861 0731 0559929501 0141 0061 0741 0251 0251 0041 0561 0179979211 0129829669769649709271 010986970989939976942931915966979935937927947927890914959948972937938908933989905967974955963865973952859935970887871909951982904940922862929932953899904902884901899875875950900885923951922961963914866914871864940909862923880872938902879860875851846832829896904864873844867846862905862875843847930834890885864865875854894819823849843883847873845898830884819875860854849833830864830853863844867833855857842826803820845826801766830767823733812781860794784762793824847795797773732794776802765847783786770730771809760799845789772787815768768820770784788728819754788791773797807805820760839756792773802771780774745775772791797717785741777813754772798746754760738720705778754761749780719763763701738775700751711721712719708706748707648731687697675771723699697739721705700724720708730682668688755704680742724651712701661707702730715734729732663696639744704689671702665681700720699686677712677736644702695705691629667671665721712676707664710718692645721713675620705685721712672731650697669663692653699707656637704692672635705680661650655697670706638672640644673698620654653683669665632655621659694612669633661645625669624610685664618683616639566644611627596599566629644696635608592650626574626617611612636596596646629631661620596591632610605614593548625604593611636602621600575575544623596603610610648595636594596573618554600588537621577604606552606604565628579570582590613593653601569597543625561558583524585614583607556566582588581589563602582600579538602551577523601569536561582651 152100200300400500600700800900>1000Coverage value1k2k10k20k100k200k1M2M10M20M# 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.

785 097000000000000086 192 32300000004 608 100000077 617 65000000281 337 9060002 243 305 62400000510152025303540Phred quality score0G0.2G0.4G0.6G0.8G1G1.2G1.4G1.6G1.8G2G2.2G# 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).

100 %35 906 289100 %0 %

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.

99.9 %35 895 99899.9 %0.1 %

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 %10 2910 %100 %

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 %17 958 97850 %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.

98.5 %35 382 69698.5 %1.5 %

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.

56.8 %20 393 52656.8 %43.2 %

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.

1 626 47320 0668 46927 9199 17810 07624 74217 3927 26727 52011 89113 72829 96016 5307 43721 8337 2239 22715 47514 22715 65826 30116 70945 72857 758106 4135 270249 1755 72913 31612 46411 1214 96521 4844 1556 31811 86412 2584 11226 509376 61914 83615 56926 22019 48830 94023 44429 90839 17370 21880 43056 369224 0908 26448 96725 5199 62265 3696 3654 43332 529 039051015202530354045505560Phred quality score5M10M15M20M25M30M# 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.

99.97%99.97%99.97%99.98%99.97%99.97%99.97%99.98%99.97%99.97%99.97%99.97%99.97%99.97%99.97%99.97%99.97%99.97%99.96%99.97%99.98%99.98%100%99.98%0.03%0.03%0.03%0.02%0.03%0.03%0.03%0.02%0.03%0.03%0.03%0.03%0.03%0.03%0.03%0.03%0.03%0.03%0.04%0.03%0.02%0.02%0%0.02%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped