European Genome-Phenome Archive

File Quality

File InformationEGAF00002868984

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.

86 513 863176 039 106273 713 951351 208 305387 918 339379 384 223335 210 701271 580 864204 293 121144 256 20296 295 85461 357 00737 579 17622 322 84413 031 2477 577 0804 471 6422 730 4591 772 0751 243 015929 168741 808616 688529 081464 169411 498363 979327 603297 071271 347246 273225 672212 705198 850184 300172 222159 907146 984135 499125 013115 159107 92298 75992 24285 12878 91573 74168 25364 58160 39457 38553 40850 41647 05244 21042 57440 13738 25436 51635 11234 14532 54031 60630 43529 55728 23526 99926 34225 65824 43224 33423 29122 34421 84321 13520 66220 29019 42418 80618 71018 07717 18017 25916 64516 37015 81015 08214 67914 34513 84713 86313 49412 90712 84512 45512 25812 16811 71411 50911 25110 95210 79410 76910 24810 21610 40410 0769 9009 7139 4499 3149 0548 9708 9888 6288 4108 3348 1408 0617 9828 1798 0698 0258 0697 7877 6937 7857 5657 6217 3267 1486 9006 8326 3946 6656 5276 3176 4906 1516 2176 1126 1196 0246 0696 0645 9625 7475 5555 5475 3645 3745 3455 4145 1865 2355 2845 3475 2044 9384 9744 9914 8804 7374 5254 4554 3764 2714 1394 2664 2754 1074 0484 0804 1534 0893 9073 9973 7493 7553 7533 7673 8153 7323 7463 8713 8183 8083 8143 6363 5463 6453 6013 4583 6143 4833 4133 4343 4293 2213 3123 1353 1473 1693 1583 1483 2173 0033 0822 9933 0163 0912 9793 0292 7692 8132 9032 8212 8042 6902 6962 7212 6252 6822 6312 4942 5102 4932 5012 4082 3342 5382 6272 4982 4012 4802 5152 4042 3702 4122 4012 3712 3312 3522 4192 3492 1742 1372 1832 1412 2402 2202 2202 1652 1342 2542 1542 1612 1242 0322 0161 9661 9431 9511 9361 8701 9321 8731 8741 9631 9831 9271 8362 0141 9221 8661 9491 8991 7981 8221 7331 7701 7141 7081 7331 8161 7651 8271 7801 7931 7391 7821 7451 6801 7101 5931 6191 5811 5901 5721 6451 6071 5991 5201 5951 5681 4281 4471 4651 5001 4051 4031 4361 4931 4251 3591 5071 4871 4121 4311 3771 3471 3611 3301 3421 2831 3031 2841 2511 2501 3431 3061 2351 2261 2781 2541 2091 2881 1261 2211 2521 2331 1561 1401 1031 0941 1191 1701 0961 1211 1631 1111 0571 0991 1051 0481 0261 1031 0831 1141 1121 0581 0731 1141 1031 0621 0911 1101 1111 0281 0751 0871 0801 0011 0341 0639591 0509759821 0801 0149759861 004950893986928970934982960931937930981881887926875890900864881888937889826898782811889829828839831849855771746873794802808779796797774839851789839806756776756757759743716723699716746748659703722725709735675687703682662629634681708656653661636632630682677649677597670656656632587628579558534598625562598563616640726636606597564550566606526546565526509601609549619560549543587505546477491510539536516526534556547491532512544504492514495508535513526489536517499541478540595529533485483542545523491476493521558543525496539528565476496539512564507524526519519538506475509482503492526513508497474433488444482509451484471490461502472485471478465460493458497458514468499466465475465456464457465451434456481498458458396465459431437424431402433442416407387451459483453435399441404408439376432440457412418430426418433397430411447408417383385402417448433386452362409430387394417410402435411427438417410416400430419423404392388351401371408360398395394372406410404402410354384341389394411360370333377357344390397388404364391328365346324327374345348372336344356365366358325367333320356355310328372356335370351386378350368373347380350397373362369363362385377373369355358332354340360360330366333312312323343327277342304357298329318316288306316342342317332319311331334317314286331292346305324313293309324338335317342336317356335313322311336318287296274295333316308306438271356310303322304305302316322309322343327331332365361378381367383323375386396361327347363364319346352333321340331334337333339314302312310270323332332314331341305318332344319323314300276313286327316305327301293302323303255287284270285280299325297301277281294278289274281261282293281279256272248257242281293259293239262242255254252277293254290269261277302272299249288265285271278276238245254305289283298253249245263274256271257267267339264289251281295283286293296298292294288299278296262280294286293260 374100200300400500600700800900>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.

3 497 978000000029 365 578000827 978 540000000000476 858 3750000544 103 77900001 179 483 47200002 489 622 05600013 496 527 69200510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G12G13G# 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).

99.5 %125 542 52099.5 %0.5 %

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.

99.4 %125 390 90299.4 %0.6 %

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%).

0.1 %151 6180.1 %99.9 %

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 %63 070 98550 %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.

97.6 %123 140 69897.6 %2.4 %

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.

7.5 %9 455 6637.5 %92.5 %

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.

6 098 015120 08071 753143 158106 102110 975123 136174 38677 335131 37560 43953 81473 09183 78742 202101 70873 57380 65197 701136 898145 685149 556179 750141 580229 667373 57422 433638 54234 09932 58363 70669 49332 33884 20634 63334 61354 94575 58219 312112 0861 594 72476 19573 184125 213103 780197 011168 240269 147414 55648 89166 33755 62570 19033 26762 73263 84845 413170 69947 69597 514113 002 309051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M# 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.

99.88%99.87%99.87%99.88%99.87%99.87%99.88%99.87%99.88%99.88%99.87%99.88%99.87%99.87%99.87%99.89%99.88%99.87%99.89%99.87%99.87%99.88%99.92%99.86%0.12%0.13%0.13%0.12%0.13%0.13%0.12%0.13%0.12%0.12%0.13%0.12%0.13%0.13%0.13%0.11%0.12%0.13%0.11%0.13%0.13%0.12%0.08%0.14%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped