European Genome-Phenome Archive

File Quality

File InformationEGAF00004177014

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.

10 4678 3516 9006 0275 5104 9744 3534 4203 9793 5983 4273 4053 1733 0062 8622 8902 5912 6292 4712 5372 3392 2032 1832 1391 9701 9562 1602 0081 8761 9021 8391 9811 9221 7331 8301 9171 7951 7501 7221 4821 5001 5441 6231 5991 3801 4581 4641 4371 4831 4831 3541 2601 2981 3241 3661 2481 2271 2601 1531 1291 1581 1871 1031 1541 0781 1201 1581 1631 0331 1381 0761 0299979289769069431 000968804933931885863869759830833787780679804718725728725645681660684716712704684699661651667649609611595565582605609550486556576525531531493498544523507516501487452485437438479493493416430447420414477504434466439414421421382398388398379379378432428388405435400402395390427407399389386379407361415396405349388376411355383379339353347375388331316370362418339339375346326343341320377355353307373323341310359334313322323310319306309293292310318343329309307335321320328314297347323345315309293249296310271286334350311300311299329287320319306317267278302258290259304255247310261302288268265311293284266318343327288294318318313303286286295299282281316287295327307312312282285279317293312351296301291323324298317341319298297307312275291270312321296311298318297302292311275314287301325277331302265276285291258267259294275254253275298295292309245260266232310262287298305298286298271317320273281292278288249294253257258260268248248272292290240257249281243307254241253276253268281253305266256254275260279280265268270220255273262228233230239242262257235242231246261278291273278303230254254227240258248230265256261257244264276275274257204242257233219237216232206225224220230211245230217230217220229217244253238265229219223232199222219217204215223210199187217211209201204197199208185218190210188203190212205191181198177184205181187180178214179215200199190196215190202189177169196175179195199191184200170171148180169155162162186159162174140164161169172150173160148132164136163174141161174174173182152154166170143172157167164162164180168157160172180157164184164164179147191153158187155170176169153154155146150139164138155161130155119143162136134132128124135134118129121128127119141136126137144127140131121143132130134133157120160129146142140117112130128139118100144112121118104124136124108127127122138147133121122139133130101115112122951051091211151301331171061421321271041111431018711112512611013010411511711811711511110212010611210611710495116891271101058790105104108113106989310299108959783898878102999897959898110901021009694100998699929692826480938610810397968999103100107102919596103888896731029584879510191107708894987587817490941058793959097100979181871058197808868941021009510791907889888168789290928686959363757810299798384887865878380104757587767664815793717475697570716972587878797468676175847980787476626965696965627065508762607662737463707765687274807583767058807755775662596570607158525758566362685378655478677661615870716963757356617063577573686071706067836771565067677665604460596244736763686468606866725458706366475655299 721100200300400500600700800900>1000Coverage value1002001k2k10k20k100k200k# 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.

832 24401 7120000000000038 830 3100000008 601 3390000012 470 526000079 985 6190001 171 983 0030000589 35900510152025303540Phred quality score0G0.1G0.2G0.3G0.4G0.5G0.6G0.7G0.8G0.9G1G1.1G# 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).

100 %13 680 147100 %0 %

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.

100 %13 680 147100 %0 %

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 %00 %100 %

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.

52.6 %7 192 57552.6 %47.4 %

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.

100 %13 680 147100 %0 %

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.

0 %00 %100 %

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.

8462561942610728469311 49914125331102730637469612434579 942578141193498773503 3383 1446003 2213363851 2924441 0573535214813 648 54315202530354045505560Phred quality score1M2M3M4M5M6M7M8M9M10M11M12M13M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped