European Genome-Phenome Archive

File Quality

File InformationEGAF00002057477

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.

528 669 360644 196 824567 352 569401 206 868242 068 158129 352 92262 889 09228 619 93312 456 2925 436 3612 468 3351 259 807757 506530 240400 851322 847264 886221 428191 246162 207140 402120 623106 16994 09881 05972 81366 01658 48553 75449 19046 49842 25239 84037 42933 79832 25830 45929 12526 65625 23023 59622 41721 65321 05520 22219 59118 61217 44917 00116 72615 53415 14614 20113 74912 80012 68112 48111 86411 93111 27010 88210 91010 39510 15910 0999 9569 0238 9429 0928 3218 3567 9808 0497 7307 1417 3817 0756 6936 4316 1776 2916 0935 8445 5955 5225 2415 2625 1685 0475 2165 0764 8744 8064 7984 5524 7374 5754 6254 4564 2994 1434 1214 0684 0524 1353 9003 7583 6663 6273 5943 3943 4863 7903 6903 5443 4693 4573 2533 2323 0672 9312 9532 9692 9682 9472 8592 9903 0212 9862 9712 8562 7742 6722 6702 6422 7192 5512 4982 3042 4562 3272 3402 4012 2372 3332 4022 2772 2572 2122 1952 1522 0052 0012 1431 8431 9601 7691 7531 8471 6781 6991 8421 6901 7001 6591 6261 6411 6791 5811 6951 6761 5291 6041 5941 5211 5381 5601 5561 4651 4201 4201 4181 4811 3391 3111 2921 3651 3681 3191 3601 3051 3451 2801 2791 2551 2851 2741 2061 2561 2241 2991 3171 3771 3001 2871 2761 2241 1981 1421 1291 2031 1481 1981 0791 1391 1271 0521 1941 1881 1271 2211 2041 1511 1371 0991 1371 1071 0951 1431 1191 1391 1841 0891 1001 0871 1201 2221 1221 2441 0881 1161 0541 0811 1251 1101 0871 0081 0231 0189849468819479209358939389029999639449129759621 01996094487493089385985280682986982685377383481981881472981177479876373473576474071371067371674075077775777575268868565666967164557857161362961958263870968166863960456359161855562263570765663665167561359862259752455557660563653153755055956361956655060650848754052154051558954857749855448950450651949845245444646947545648547145349748748549251349948749244245444048045743442048046848348344548245246243445741946944546642643646445143544045146241143741344745043746645653547761546343146044247144349243645644044648543243343145945941043944344346444845945346545844850746751249145648249346950949747648858947345953949245551048346543845043046650150145846949550952952752156954749653047850349045848443042647646247243344748451044447149351545846144149647039543741243942538642238640941743937543038538744641343744337133437833137832535631935033031436133930535134331833232234933934133633833933035230431031333032731631132533035231732129930631329631333730526225229423826724226630128428430125825626329722627421523524121223122522522423425925022026426322125020722323124424522424324922924122422724324623023826624624923923720922420819825120223221223819020920520221420819218420919020719222521620519518620519820623321119621920418121418821620622322621422719617918318520619419619618918920418621520519819820019521817517518018117519320115316216818514918019119518719720819020322416818318319619720418220419724120820219819219620522720519120519522521119821219720617422319222121218923420017920720218318819619019019818920020222819218218520919318918821118217017518617716916716016717617616115115716417716615517015915317619114416917416618019317718317619716118515215817615816016814613514216916015316114414816316516815116313615218016716017517515613014615815617823615619414415015213413614815812816014215714715713914515015112814615413915714312315614514813014312612214512513115214812913314113713812414415313913314714614014815113016716217315613013214814913213013814112312412712811213412113914412713810610111311510411810911812212812411910513713612511012013094113144134113103134971119711911298111113118101100111899810313414313414211412311910312110694101881171019799102105100831171081201081111041041081151161091161181081201131111031001141141121109792 922100200300400500600700800900>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.

868 889000000023 775 349000419 841 449000000000246 280 6880000268 970 6390000568 880 20600001 178 697 6560005 745 271 31600510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G3.5G4G4.5G5G5.5G# 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 %55 760 23199.6 %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 %55 586 68099.3 %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 %173 5510.3 %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 %27 988 69650 %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.

97.9 %54 828 66297.9 %2.1 %

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.

8.1 %4 544 9108.1 %91.9 %

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.

2 164 68647 58730 44658 46044 42446 01155 74170 83030 93354 84824 75321 35333 74733 22418 31440 91126 16229 97545 23659 45066 98059 34671 11653 45987 199138 3329 024261 83613 09612 90929 93926 36112 69532 42813 93713 83325 02529 5568 13445 062736 61332 10731 66650 27743 26977 89169 904100 244175 17719 29926 71023 06531 33916 32727 12028 49221 31174 45622 28743 04050 521 972051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M45M50M# 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.

99.68%99.68%99.68%99.69%99.68%99.69%99.68%99.68%99.68%99.68%99.67%99.68%99.69%99.69%99.67%99.69%99.68%99.68%99.68%99.66%99.69%99.69%99.41%99.59%0.32%0.32%0.32%0.31%0.32%0.31%0.32%0.32%0.32%0.32%0.33%0.32%0.31%0.31%0.33%0.31%0.32%0.32%0.32%0.34%0.31%0.31%0.59%0.41%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped