European Genome-Phenome Archive

File Quality

File InformationEGAF00000488018

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.

63 843 89042 775 21219 808 43511 590 9574 788 9713 248 1361 361 2131 023 140470 973366 996198 587162 158108 03592 34575 97068 03160 40555 94252 14947 77745 62643 96042 37840 80639 54938 22537 08635 63534 33033 66832 85632 26831 71531 47930 75530 00329 73029 35929 36128 94028 40027 98927 36427 49826 92427 29026 06726 03225 34624 92024 96824 58824 33823 64823 53523 04822 98222 76022 80222 49921 95521 84821 75221 82421 54921 06520 83620 09519 98419 71919 79919 86419 09118 94018 45418 68218 39317 84317 63817 83017 45017 30017 35017 20016 84916 78916 31816 45416 28115 99715 57615 66115 38515 52515 10614 75214 56414 56013 97313 80113 56213 50013 11113 24712 76012 48012 20712 34911 84811 87311 67911 56611 42611 24910 95810 88510 52010 31610 0889 8939 7939 5769 4589 0829 0738 6708 5028 4958 1948 2268 0177 8717 7837 7427 3637 3907 1506 9066 7156 7876 6386 3386 3806 2576 1676 0435 9155 7515 6115 5445 2415 2695 1515 1384 8784 9704 9144 7284 5454 5274 3794 2224 1834 1653 8783 8993 8803 5953 6943 6033 3723 3683 1913 0473 0543 1252 9583 0482 9482 8712 8092 7192 7182 5432 5792 5322 6252 5012 4842 4392 4352 3232 2732 1882 1352 1351 9881 9622 0001 9791 8631 9011 8741 8171 7471 6841 6591 6891 6591 4731 4831 4531 4731 4101 3111 3531 3901 3341 3051 2811 2431 2181 1681 2211 1511 0441 0021 0179029629369629038758458988388297797828047547657837007307437527186837286566056716466006085815685955556085755395635525224975074685285114514484464314284304003984334203993703673383593453673573383113323623172923022982582753002982663012612432512492412482362112192311972292102032072082341992082191971871761771921691671641881521991711651781501891571921791601721791621751611811741741701841911921741891721701481601651481361361331191421291281321381031311251281271421399913213814912011312414613713112213014114812113012713112210911611612111412411212710311299105889497102871081081039183918810592758793848780848779108858485929388938610498839610474838990676481507363556164616947516359486565605664455658524348454653394545443246463829374529353750363437292739242533223022322727222329211724271639242520232017282021191622252021253124262220232128162126261326241721152024212221172319192520332230242419302414222322162114191816221815141810182121191817182326241519222418172421221623141721152313202122192217101621182217161119111116141416181711111215131378891914119141214101271512121412161191395811911910981098729792979355733452646233337224232211341324312322223112132123524112231312154342333314432352333314527410102576775232222838222316131111100200300400500600700800Coverage value1101001k10k100k1M10M# 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.

498 03700001 116 517459 9922 928 7071 567 338441 5751 154 029442 520450 868659 127279 410935 279609 248823 0171 467 817707 1801 387 587791 5441 324 2092 006 4202 020 3082 368 8653 978 5723 637 0063 414 6243 973 6718 375 67213 618 8728 493 03414 410 40829 431 15241 976 47721 480 02556 293 80638 413 31266 058 73276 296 553148 822 14000510152025303540Phred quality score0M20M40M60M80M100M120M140M# 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).

98.5 %7 395 14398.5 %1.5 %

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.

97.6 %7 324 55697.6 %2.4 %

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%).

1 %70 5871 %99 %

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 %3 754 09150 %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.

94.6 %7 102 89494.6 %5.4 %

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.

39.1 %2 937 38939.1 %60.9 %

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.

375 7632 3999674 0121 6262 2173 9323 6713 97822 93711 9779 27717 7429 2365 15238 2597 06618 67212 1883 38922 0242 30811 09551 2482 99114 7682 1401 7841 745507 3422 1111 7782 0802 4961 9022 72045 659324 8994 6162 6466 3505 5963 76012 7486 8648 74227 44014 23813 57817 41626 41611 35630 35222 16828 20042 05671 0705 597 020051015202530354045505560Phred quality score0.5M1M1.5M2M2.5M3M3.5M4M4.5M5M5.5M# 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%100%100%0%0%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