European Genome-Phenome Archive

File Quality

File InformationEGAF00002337370

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.

172 285 482279 727 717357 730 670390 462 176377 221 345331 743 908269 819 211205 807 337148 752 357102 890 43768 557 99344 234 14627 885 98217 291 94010 599 0036 479 8643 981 3782 507 5661 610 7701 090 267770 918571 004448 276362 134305 066260 101229 296201 822180 376163 011147 851134 732122 362109 29899 30990 78682 55876 13771 44565 82360 38455 85951 86547 16144 67540 85538 81135 75034 46132 31530 23828 41527 48325 92824 81823 25122 47321 98120 56619 55918 69618 25917 20116 65016 17416 45515 05714 38113 95314 09613 34012 82913 05912 35012 57011 79511 93011 24111 23310 81110 56810 2909 8599 9909 7319 5189 3759 3629 1938 5068 4567 9697 8617 8837 5467 6127 3177 2827 4507 5826 7956 7836 7476 7316 5756 5966 8116 6226 3606 2596 1426 0315 8855 7305 6375 5365 5915 5055 4785 1195 4265 5305 1154 9214 9794 9334 9624 8234 8644 7344 7214 7894 8214 9334 6534 4604 2834 3084 1823 9903 9464 0024 0373 9603 9183 9893 7243 6173 6033 5673 5923 4833 4343 6273 2893 4633 4763 2893 4263 4633 2833 2313 0133 2903 1843 0393 0123 0423 0072 8953 1362 8773 0652 9202 7522 7912 8922 8842 6852 6912 7682 7212 7092 6362 5102 5922 6032 7082 5022 5922 6292 4692 5852 3502 3652 5462 5182 3502 4822 3702 3472 3362 2482 3602 3122 2502 2052 2992 3202 2782 1522 1752 1542 1252 1362 0082 0242 0612 1352 1762 0962 0582 0901 9782 0461 9941 9111 9871 8871 9171 9672 0562 0402 0302 1262 0081 9531 9361 9031 9522 0241 9681 9621 8641 9962 0881 9471 8771 8101 8631 8121 8361 8001 8861 9231 9731 8501 7981 8641 7221 6171 7231 7901 7261 7211 6341 6021 6341 5771 6161 5851 6251 5721 5301 4831 5311 5821 5591 5551 5361 5651 5401 5731 6361 5291 4071 4921 6491 5961 5181 4661 5451 4711 5701 5341 4721 4441 4921 4081 3651 3631 4551 4271 4351 3251 4221 3671 3951 3251 2871 2841 3221 3851 2761 2631 3331 3371 2111 2421 2231 2601 2721 3011 3711 2251 2791 1781 2301 1861 2111 1861 2191 1591 2231 1471 2231 1411 0891 0971 0891 1071 1571 1141 1421 1621 2021 1781 1531 2291 1641 2051 1521 1081 1541 1151 1251 0721 0359419841 0131 0131 0021 1021 0301 0169771 0099709901 0169299769459999619919089449341 008949876928914883855942834911959876908880957887843796808793789792756780824799749752745792771720723789769732832745648763775784764746729736698732699665647683695685708709665688662649688732676640685700676729626678608676636595612597548594602629570654640587599569578587555518577604666544559624577550572592556562504550569537576535549578536583562594599592588574600584600603662573615570542578538590589618589563598602623572590606593605554565563535527533519500490523493497462510540559512482487498477467521478448484468441496508497468536514490458450457455466457510519499519499487462499474448451461460442452467467410441441456414426457440455429480439425419402425433403440377392367435411406407428388453426397412362408419415421394404392389420388417385354405373440398373415376374386366413407423359382425379387351368318375360299320348345322356367384355386350342287353337333348312336330312382355361329352358316354372390332349367309345330342335311328357312341342318308287317326301341349315282329359303323339327321301301288309319332317317305332321314328329285336339322302318327337308292320314285310298334331325287321297290332279335330315344324316319287305310311289288283272274272262253270276295259310284281249293251288283275319279293248262267277251244233225248224247247231234231245259267272230318279234266227256263246258261268264249258224256254260254233227231248243217272235239227226243226248227237221248239231219222223246214215254219217226203252229220219234216239197223195208229208175204193209217188199179196203214203220196277263207197203208191197189194186202182190190200194202193190207192198184187192172206202205197184223201196185198194187204188198183180179170208166186177207185172172190193189217194156182204190204182188176161180195192203194184185179173209189180185196183165184182189199186167196165178171159191152179163154170213194184190167159148160161161161171174155174172180197221 457100200300400500600700800900>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.

2 784 3590000000143 171 1000001 128 651 997000000000628 596 5390000682 415 60900001 179 009 35200002 196 149 5530009 097 931 74900510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G# 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).

99.2 %98 891 85599.2 %0.8 %

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.

98.7 %98 434 82698.7 %1.3 %

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%).

0.5 %457 0290.5 %99.5 %

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 %49 863 27950 %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.

93.7 %93 394 95293.7 %6.3 %

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.

10.7 %10 708 84910.7 %89.3 %

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.

7 374 760141 347111 333174 774147 569162 807179 283227 918168 915188 726116 61290 856100 77896 23961 168106 06276 70486 657111 083145 335152 490169 917216 553149 375216 087300 15467 046478 92275 60563 34995 113103 86391 101121 27568 57272 52185 033100 15554 539151 7601 162 51399 256108 299148 044118 606180 777154 856232 118323 69190 46590 79793 34098 46171 668183 29798 36889 450162 53895 116139 07494 173 232051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M# 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.

99.57%99.56%99.57%99.58%99.57%99.57%99.58%99.57%99.57%99.56%99.56%99.58%99.58%99.57%99.56%99.59%99.57%99.57%99.58%99.54%99.58%99.58%99.65%99.45%0.43%0.44%0.43%0.42%0.43%0.43%0.42%0.43%0.43%0.44%0.44%0.42%0.42%0.43%0.44%0.41%0.43%0.43%0.42%0.46%0.42%0.42%0.35%0.55%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped