European Genome-Phenome Archive

File Quality

File InformationEGAF00000488026

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.

139 014 57945 522 44813 364 3015 290 2901 855 901852 801399 608242 112165 613131 688108 97297 19288 64281 37975 50870 52966 54062 62060 31258 02555 75953 83251 82350 39149 33547 71145 58145 44344 00042 19240 96439 93939 74438 36936 78036 40736 47334 94433 96233 40432 34331 98231 23530 44329 50029 12428 54328 23728 21327 61727 32026 81925 96425 55324 77424 61623 94423 44423 02122 90222 29921 73521 64521 01720 69520 15619 67619 17018 69818 23818 12417 76717 32216 90116 41316 12015 67015 37715 19114 82114 56914 07113 65413 46613 38613 10013 06312 60912 20311 81511 60811 40811 16110 95110 79610 44910 21910 2189 8309 7229 2649 4789 3089 0338 9778 6148 6438 3828 1697 7927 7557 6867 4257 3647 2627 2346 9316 6466 5666 4086 2046 1656 2166 0945 8815 6965 5975 4785 3545 4655 3195 0974 9414 8034 6984 4574 4864 5864 1974 2134 3074 0074 0453 9753 8323 8093 7243 4533 5183 4563 3363 2153 2003 1673 1062 9823 0042 9272 9552 9042 8252 7582 7282 6872 5952 5262 4202 4122 3722 3082 2692 3142 2932 1612 1592 0902 1321 9691 9642 0691 9722 0101 8731 9531 8761 7901 6551 6271 5891 6091 5741 4801 5041 4481 4371 4111 3481 3911 2951 2981 2471 2461 2141 1961 1371 1201 0221 0441 0529609699519729469919049479149028498769118408218088317938587627187267267207276646696816256466125655835485515805575345125345055505295215734894764494614884494503974334714264664173994063943594204073873193173603193143453463062943033293063012852672723032712812532742522402582672702502392342512182492342432162011911671701641701761751591711601461591591691471571481401481441281491511331181281231241421381281141321461181471221181221231261011391311211081171251161271171211201259810613083102110120115115109861008774939295909010273918588898680847768857865837066696364706166765761566363605362676645546163604951505963666046767366697482767661746469557055636259585771685556544443454160494237495436424940344862524744433840385448494655474142414840614846536353525254514541525760534344405854334845424140314524353544395529344150484954454151414136464654374536413139433229363336242229233423292725312631383528283843283531222926232027211822182524132220232323262823252821152320173020232416161925111315191614813131081459756614168131210112161071053694484758428732343332224213225523152725332311111212211211121111241346325542321253544112311212416331321123143311341114122222321111312111422111112113422665443531197232351521333413213112221421122412121111112122331511111214111512122175413113121100200300400500600700800900Coverage value1101001k10k100k1M10M100M# 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.

443 7200000942 707401 0872 537 4251 426 064417 7601 020 308390 533396 443605 430250 756828 025560 929735 8121 316 399644 6111 209 082704 9281 211 7481 783 7421 829 3302 132 0773 567 4173 297 4383 113 6823 565 7167 542 03012 276 3817 640 76312 874 34626 468 69236 880 77119 406 22550 859 32235 105 68959 934 20370 007 658135 913 45100510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M110M120M130M# 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).

98.7 %6 714 85198.7 %1.3 %

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 %6 668 40498 %2 %

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%).

0.7 %46 4470.7 %99.3 %

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 %3 401 61850 %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.

96.9 %6 593 15296.9 %3.1 %

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.

11.1 %756 14611.1 %88.9 %

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.

285 3441 3336842 6551 1021 5092 0922 4802 42715 8477 8935 12912 4775 4303 19527 5854 59412 8828 5182 35016 2881 1068 14338 6731 3726 8531 2271 1801 311371 9501 5551 3461 4921 9521 5242 25839 819203 8033 8802 2745 2504 8163 27410 5765 8907 57823 40212 24012 13015 24422 5789 98627 01819 11425 07837 30064 1725 384 058051015202530354045505560Phred quality score0.5M1M1.5M2M2.5M3M3.5M4M4.5M5M# 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