European Genome-Phenome Archive

File Quality

File InformationEGAF00002307909

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.

397 183 376532 222 619536 958 611447 086 288322 299 326208 127 688123 368 59268 332 81336 073 27118 430 0269 282 2554 735 2422 502 6611 420 106883 489610 334454 517359 082299 953252 355215 772184 923161 753140 503124 054109 50596 90686 75977 79170 64264 25958 63354 51149 18846 02242 17240 02337 00935 42232 77831 19729 88528 16526 07525 71424 09922 87422 11420 62120 29419 35418 18017 90416 61716 38816 33515 83415 11014 42314 52113 79813 32912 46012 36611 89311 62811 62911 62210 75010 38310 2839 87810 1389 7909 4609 4509 3558 9488 5548 4608 4728 3317 8377 7727 4477 0877 1016 9356 4336 4646 4246 2146 2995 9536 2576 3676 0425 7895 4055 2655 2235 4624 9694 9454 9984 8234 7524 6374 7234 6464 6504 4464 3514 3034 1834 1543 9764 0073 8693 6933 7463 5483 7103 4023 5843 5253 5783 5093 3363 2573 3083 2363 0833 1933 1303 0762 9872 9982 9763 0032 9953 0062 7912 6972 7092 7212 7482 6672 6392 6392 5482 5022 4772 3792 4242 3452 3022 2852 2622 1852 1742 1542 3082 1062 1042 1942 0852 1382 2082 1202 0682 0392 0291 8711 9011 8811 8031 8601 8261 8091 7401 8491 7901 7951 7771 7981 7691 6881 7151 6831 7211 6771 5931 6101 5821 6561 5731 6911 6081 6001 4741 5541 5571 5521 5901 5191 5201 4971 5281 5171 5391 4591 4481 4261 4581 4471 4011 4091 4631 4581 4481 3851 4451 3471 3521 2941 3361 3341 3611 4191 4581 5521 3551 3911 2891 2881 3021 2911 3101 3041 4171 3111 2051 2211 1531 2131 2111 1961 2361 2881 3061 2811 3261 2441 2561 3541 1721 2131 1411 2351 1641 1851 1361 1861 1771 1921 2561 1761 3221 2251 2531 2511 2391 2321 1441 1441 1081 1851 1601 1401 1881 1111 0971 1781 1491 1111 1281 1091 0891 0421 1061 1181 1721 1821 1631 1341 1331 1161 1171 0401 1121 0851 0571 0331 1021 0491 0271 0311 0181 01095294387589586292085386986884583689986183586586284785182290581783276684786989979578280374669473275976466678469363674070271670568269368772271273665467470068068465666463662065367264361172062256759660561059862155657356858557558461255556456553652756751753752049952649152249648447551748448551444250942846346253954150446448448346547745844844447444447144648545243346444645646148945947748048340546145542144839547644042338843444740440338844742843039041441641338440940837539542043038837236236436737337736538936537841939338339536536534935931736934433934334135036538134135437934033531535037234233431937536837331835333732834235235733539138132535434437733833734835733234737735635935434330934735435034130834735033031933936236736432530635935035735936533434638533934235537338134133633832333136233534434236936532532837134734034531333532729331732730533432331832731233833236434337031636438839934236234934836334632933129734936132736831134633330933131333431731732634732931536732730831831434029832831431730731532028629831028627729329125329928628728430430829926526829728525930531727430627330430228028232431329928428130525626927430126130229129830027526530128533828328330324326929329830828228825725827928726028529424224724124728623425622024521321224022022824619023122121522222622724923121420618221523622222618122019222020220222621119818819520420317119320520019517316917320620317719217918416617617816418017014816617918216016115214815016112217413917116617214816717914516216216514516215316816215916816816615914214515915414212716512313915414615314014917516514513211813413013316214014513815014815212813915714813014213514715811513012611612713212815513414513012112316314413613612711714014116914514413212514810915513911011812311011911512112612712613313411212312012413312811912916713214715513314111214914913513714014814114911812712313412912814915713413313311011912914312916114313414715513316816015712113312613611513014111511310614312311213211412813214813212612014013712112013211313314212112314613211713016612313512014310912613911913514013711112711912110112311713410812096116109108101123110141113114102102114 501100200300400500600700800900>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.

908 320000000081 759 867000885 515 780000000000478 536 8270000512 013 6520000879 315 45100001 644 628 5200006 005 890 60500510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G3.5G4G4.5G5G5.5G6G# 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).

98.6 %68 514 11998.6 %1.4 %

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.

97.5 %67 749 88097.5 %2.5 %

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%).

1.1 %764 2391.1 %98.9 %

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 %34 730 36150 %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.

95.7 %66 477 22295.7 %4.3 %

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.

6.9 %4 765 7766.9 %93.1 %

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.

3 688 28859 81237 36973 38953 61955 19577 83385 68038 59365 09230 88326 07950 53341 89021 39451 00735 30138 04960 65266 29769 14370 88685 67367 868118 614180 17112 278323 59117 46116 72141 26634 35317 01042 13717 55417 52137 07236 65110 96055 920919 03346 91954 37671 85065 174111 91095 831152 130202 56734 86939 69639 60646 41332 65453 97050 15242 125103 94045 49670 06961 733 523051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M45M50M55M60M# 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.

98.87%98.88%98.89%98.91%98.9%98.89%98.89%98.88%98.87%98.86%98.87%98.88%98.9%98.88%98.87%98.9%98.86%98.88%98.87%98.85%98.89%98.9%99.15%98.79%1.13%1.12%1.11%1.09%1.1%1.11%1.11%1.12%1.13%1.14%1.13%1.12%1.1%1.12%1.13%1.1%1.14%1.12%1.13%1.15%1.11%1.1%0.85%1.21%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped