European Genome-Phenome Archive

File Quality

File InformationEGAF00002337303

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.

145 557 164242 987 965322 906 278367 494 327371 417 282340 916 417288 788 390228 914 360171 143 848121 791 80383 188 63154 916 62535 215 61522 117 82013 647 4188 344 4325 125 8213 206 6092 056 2061 377 174968 490711 420549 364450 867376 652321 447278 768247 741219 482199 670179 667164 127149 265137 466124 443115 179104 40696 64188 89982 62677 52770 76366 01061 36358 49754 14750 39146 81942 40740 53037 27635 14633 33632 02530 28627 68726 45024 96923 58223 08922 48421 46920 74919 59718 47517 83017 31217 18716 04315 73215 29915 08514 48113 64113 22413 19712 61112 43612 17912 21512 05111 72611 42811 03110 94210 33610 1909 8259 7609 8989 4439 4739 2359 1258 9708 6328 3728 3548 1908 2687 8737 7767 4607 3577 3447 2547 2237 1897 1976 8556 9846 8206 4536 5716 3436 1705 9935 9815 7495 8025 7675 7485 8065 6485 3165 3625 2265 1774 9684 9504 9165 1764 8424 9094 9484 9444 7254 8224 8224 5714 5034 4614 6284 6354 3454 1684 0844 1284 1284 1804 0894 1163 8303 6613 4873 6853 5783 5353 5843 4843 3273 6023 5183 3253 1573 3663 4683 3983 3763 3903 3333 3313 3693 1603 1062 9853 0173 0342 9902 9272 9932 9062 9742 9022 8632 8212 7602 6232 6842 8512 7472 6852 8442 6262 8182 3992 6152 6612 7272 4562 5352 4752 4162 4262 3822 3232 3612 4172 4402 3012 2532 2592 3052 2622 1342 2082 1902 1572 1482 2342 1582 1702 1202 0972 0642 0902 2501 9332 0461 9932 0171 9381 9832 0171 9292 0372 0781 9251 8961 9251 9552 1381 8441 9521 8571 8891 8651 9001 9902 0081 9601 8751 8071 8631 9391 9231 8811 8821 8891 8971 8221 8721 8531 8821 7421 8231 7861 7241 8551 7121 6991 6061 6381 6271 6781 6891 7261 6871 5511 6171 5721 5641 5871 6261 6381 5611 5801 6461 6061 6321 5901 5391 5121 4491 3861 5401 5501 5661 4801 4401 5081 5401 4031 4551 4641 3451 4051 4831 6011 5511 4701 5081 4441 5591 5101 5581 5871 5291 5091 5111 4581 4701 5391 4671 4401 4401 4421 4641 4721 3771 3961 3691 4681 4831 3951 5551 4441 4361 4061 3481 2841 3171 2671 3131 2901 2781 2391 2381 1651 1641 1421 2011 1251 0901 0731 1041 1361 0581 1011 0931 0151 0261 0581 0451 0811 0711 0431 0901 1341 1151 0721 0851 1251 2781 1201 0921 0131 0629991 0121 029935962907913916962999876950827862932966948949905855903927887924859925906851924938830913885931896855867801854827885813861863809835836835838797836848834811831880832894905845848832767820863895926869804780751729769790768782751751789784732728727692705673674699687699661646704654642661695704656595631680651668667613647684661666606643640607610636636631662663656599609625601588627623552621565565601619613573607603568583579561520611566603591554544582557586548602593536596549498536493545535546491548494497486502513466470491455449481454439459527416474479457513450470501511513503497506494472463523543485504486453447460457434459433451449505440453452443462416461478464488436451442464466443458431524441443445486434470444457452446416460450438451424428432422452462444419435443411381416452414404443377349352411405372406369419423415420439407385432403404361428418382388366388407390387391392397370373365366377344358344313349351366342334339361368378331361372362375395357384373377387313351390340317309334319332316308329327314344315323354313326333344291315333289318350304302286294334321325330298324340312338305320321300328306332302317328282272350301340296304301295295304299320323285299285292274276274284289287283267278266290264289274252289250269277336295305266252258282268247275278272279252288308256238258240276247266265267265240263269234264233272233266257253256263269291286271293273280252272301276274283256259285249276270271288257241337261267237267256249276260249287242251244243235244239254224211225225196240227245270214247231222216248249222235261218221243236248241229232226231245255226213206221213228233278248228225199187232191204219203233208190205214194187187192188188181172199179194195183188215211205203189205202174186191203194224179198192194201184205187198176195194185171186224180241196179187195176202204197208190175204192181165173166162153168161161174129181165147158151162182243 037100200300400500600700800900>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.

2 957 179000000057 716 326000781 532 059000000000445 525 2550000516 539 51300001 062 484 87900002 149 595 49400011 113 871 86100510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G# 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).

99.4 %106 207 40899.4 %0.6 %

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.

99.2 %105 928 16899.2 %0.8 %

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 %279 2400.3 %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 %53 411 33350 %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.

94.2 %100 638 03894.2 %5.8 %

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.

8.5 %9 104 1738.5 %91.5 %

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.

7 713 795148 276116 738184 092159 144177 155186 200249 731187 430198 517122 04594 90399 21499 91462 031108 11979 03691 005109 219154 168163 052178 542236 618156 672226 422321 00367 324518 48673 58862 20490 426105 04197 112119 08367 67370 81381 276102 49652 243154 0641 169 41795 91298 982152 081111 692178 715155 447226 873367 89479 14085 95786 83593 14560 939175 47885 26391 648159 65779 344123 309101 286 896051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M# 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.

99.75%99.73%99.75%99.76%99.75%99.75%99.76%99.75%99.75%99.75%99.75%99.75%99.75%99.75%99.74%99.77%99.76%99.75%99.78%99.74%99.75%99.75%99.81%99.73%0.25%0.27%0.25%0.24%0.25%0.25%0.24%0.25%0.25%0.25%0.25%0.25%0.25%0.25%0.26%0.23%0.24%0.25%0.22%0.26%0.25%0.25%0.19%0.27%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped