European Genome-Phenome Archive

File Quality

File InformationEGAF00002405112

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.

112 823 58424 028 63610 228 7985 915 0994 041 2693 041 5912 438 4792 035 5451 734 5391 507 9531 329 1771 179 3461 060 079958 010863 232785 820712 351651 756597 638550 384509 941469 588433 853401 824374 602348 316325 996302 751283 478264 818249 263232 899218 896207 672196 787185 549174 111165 057155 974148 013139 374133 707127 526120 404114 364108 768103 35598 61995 16490 16286 75582 48378 71474 87071 65268 74165 86962 13360 26357 80755 34652 84650 72049 50447 15245 49643 63942 58240 88339 68238 42937 11735 82734 47833 62232 49031 30030 95829 97528 99127 94726 86826 05925 37724 63423 57823 20422 41121 77021 30120 50420 39220 02319 48418 81718 55717 69617 31516 58416 43616 18115 76815 38514 84514 57314 32513 90713 63313 14012 76212 34812 16511 89811 66011 50111 25711 04110 79110 46310 20810 1389 7129 7409 4079 2099 1238 9648 7438 4908 4328 2488 0808 1387 9747 6767 6407 4247 2507 2447 1436 9066 7896 6836 4526 5756 3136 1736 0055 7705 9805 9355 8405 7965 5725 6005 4285 2745 1595 2825 0695 0144 9304 9054 7914 5284 3714 4944 4884 3674 1744 1794 1963 9864 0373 9334 0083 7783 8363 7773 7843 6273 5933 6823 6913 5823 5103 3863 3353 3643 2403 1823 0953 0703 0203 0742 9952 9372 8882 9572 7892 8702 8222 7822 7372 7102 6492 6152 5592 5672 5392 5162 4592 4082 4242 4852 3072 3022 2452 2452 2602 2412 1652 1582 1582 1362 0952 0362 1022 1141 9631 9241 9501 8482 0291 9151 9761 9121 8631 8001 7521 7441 8111 7611 6691 7451 7051 6871 6001 5581 6161 5291 5661 5471 4231 5201 5271 4961 4781 4491 4581 3981 3651 4801 3791 3661 3671 2901 3291 2831 2971 2921 2031 1571 1611 1941 1891 1171 1301 1231 1241 0661 0381 1011 0611 0961 0791 0421 0439469749511 0739689769349349949319219219438468971 0229038879128899178188888709008438568368568067947897847817577638347947677697797876877326967076966947306576907136936586786807136716606676386876236966226535886456096145915875695835925915915935885525395675685655675396045425525796015495345344955315525255325015255245254844824935185204634594754814564835244694764674374634714694274354444514024324404694534524464244904224324134313964093753673543883553873803443883683313733723663153893903673763913633893953573593543813503773523564113723943533743663593463543403383193083433293553273132583462913303273783253423083803133173003263363133083203252753133003352822943083062623253012992812512712842913093213073023113223012962933023122642983092752402862872932782652912882312552742752942692612632372642732912612762352352552442492632442652502512362512552532542522402322622672512102442242492502462252472222162392632032502111972052302302042191912152162162432312042082252162032022101902352112042252192432161991981982052312132071942171751931662132051802041852082012161901851901911981761721811852001781941721982211781811931971881941911691951821951911901621771671841851551951862081701761831761622021571671812221851591991971781751851672091801731671531831701741671961891751841651551791731851691641621611761671711771711671801591571501701441651721661781741541741871781611621711691601651451611551501721711591901671911661421801501611591881571681491691431521681531591521431481451831621471471681551641391441431491401331451611431321281281221301211401371391341191371361431651551431631321461261311451371331501321341701411641551521471581401321181441331621501471291341191551321451361481481481231161241101251281341271131161381341311231181371131301371181221381251201361321361311091021141171031251241119212212713310611511011512812093941081141121081201181191031171121011018795901301051021231299612111110810410511410410111695111979295104991039910493839997108102109951127510493108989812090951081218810095881138510411097112991189791100928199758798799286112101909983997710687938598907181888696889689979976100105869490939970 193100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

0093 664 6630032 338181 9211 127 3492 765 7861 954 7864 432 798791 046394 547493 748259 2292 113 6863 787 8503 741 6633 295 9112 301 093851 0491 252 6603 034 4323 596 8434 754 1807 392 6006 531 71812 746 6507 933 3186 110 49624 707 90233 399 19121 734 73429 624 53959 454 50759 592 65731 507 907126 118 45883 538 190138 100 626160 559 340332 568 60900510152025303540Phred quality score0M50M100M150M200M250M300M# 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).

95.3 %24 824 17895.3 %4.7 %

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.

93 %24 217 27293 %7 %

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%).

2.4 %606 9062.4 %97.6 %

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 %13 024 99050 %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.

88.8 %23 123 33088.8 %11.2 %

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.

1 225 8021 2104 1102 4704 613407 5661 197 7233 99712 22264 794182 378719 90115 92171 7223 301 17213 821 5511 21468 7314 942 883020406080100120140160180200220240Phred 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.

97.14%96.83%97.67%96.91%97.12%97.37%98.07%97.11%97.81%97.78%97.79%98.1%97.65%97.66%98.17%97.17%98.1%97.48%97.53%97.97%97.82%97.41%96.4%98.97%2.86%3.17%2.33%3.09%2.88%2.63%1.93%2.89%2.19%2.22%2.21%1.9%2.35%2.34%1.83%2.83%1.9%2.52%2.47%2.03%2.18%2.59%3.6%1.03%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped