European Genome-Phenome Archive

File Quality

File InformationEGAF00002441839

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.

32 465 45778 495 853151 449 159236 689 056311 722 948356 673 203363 224 894335 019 761283 696 427223 322 114164 997 548115 346 68276 914 49849 328 62230 621 61318 617 13811 158 4826 694 2774 059 0592 556 7831 691 9001 186 255876 115683 378560 296471 795409 804359 439321 097289 899264 228247 728227 674213 055199 019185 514172 370159 168149 870141 619132 637124 511115 516107 736100 40393 74188 01681 85076 22070 96766 73462 49358 01054 98051 56448 81745 81042 98440 34638 96337 07335 39434 10833 31331 68030 91030 14028 84227 43726 61725 84825 06324 46723 27022 12921 84920 96020 34319 41019 26118 62518 04917 86117 17516 69615 96615 27315 37115 25214 96314 22113 91013 72713 42912 82912 79712 89912 65512 38211 60811 51711 33911 07010 95510 64010 3329 9359 3849 4989 3659 3689 1318 8409 0398 9968 8408 6288 3638 2208 1207 9327 9387 7967 8007 3757 1567 2017 3297 3347 2297 0927 3036 8926 8626 8326 5306 4066 2476 2606 1396 2726 1336 0886 0375 9395 9005 4045 4235 2745 5745 3005 4135 3285 0265 0124 9664 8894 9074 8014 6274 6474 5894 6494 6044 5354 3464 3894 2924 3724 2724 2214 1724 1894 1424 1064 2483 9504 0543 7853 7503 7753 7103 7423 5803 5103 3353 5193 5193 4703 5133 5443 3703 2653 4123 2703 2963 1643 2063 0393 0882 9503 0313 0183 0182 9683 0373 0413 1403 0342 8862 9112 8592 8882 8542 8662 8452 7702 8272 7382 7862 9342 7782 6732 5092 4882 4142 4702 4552 3582 5132 3912 4132 3532 4212 3112 2972 3582 2692 2242 2672 1982 3242 4262 3462 1862 2342 2272 1432 0892 1602 1431 9962 0102 1182 1282 0852 1422 0232 1481 9911 9761 9261 8621 7831 8671 8641 8331 8291 9601 8951 7851 8121 8031 6911 7471 7801 7461 8361 7961 7661 7571 8241 7451 8141 7411 7511 7231 7191 6841 7201 6601 6981 6371 8051 6921 6961 5671 6211 5201 6571 4371 5481 5101 5071 4611 4131 4631 3691 3921 3941 3901 3381 3641 3221 2211 2411 2841 2771 2681 2621 2171 2031 1321 2211 2651 1741 1781 1261 1211 1511 2001 1771 1631 1921 1501 0471 0271 0691 0361 0041 0321 0351 0381 1021 0801 0351 0671 0501 0791 0331 0279561 0411 0289869519611 0649799729229238929548919109488848939029319229639751 012956940946934879916820794894856878917884932850902838823851783823824868799853842885829843799818799847752825774828799758823864758687733730670727758806767732747697749722720666713695685722645716615657690682681654690622639631643611613636588631590593579624640614590565625575568584587625616588644604589584578592573572523560518595553576601608606617625570614564546509551559548565553549582539563605554494523540495503492496489477495494486481498493518496536553522496452522480533479503492472493517494535469459472489450463472441474458438475525451447466467524482510499447426480462467501451474436472505470483473500471441410448438445440462493523499437466412448436406445469492506441426478462429421467423426387468436437504479446413392385399408428411436422409439405388386393379381402389435428422419380449383408411378400423432378380423418412434438374365387393370359409389421349343345357376341360368324333322327327326340384398311331311314338299340304287310334334296320298309299328309309306279312303299315335334299284307327329308321275314291293300286321272280295258292324312322281272272266298297262328264296298270295296279287294288269291300322298330306323323314312285276286292250276269297303293335324350319320312351304320319305318314335325330343304309337296298297310305272311289277309280290301318335303336317328319292327282325291313300294332321318320300327317290282280275256266280255281287267273277293287260296245322264293296309283289301341281263310297287323296305282278290284273301276287271257282265267271297254263236259300284297286271270255305307289268293297256265246254268235279230277251263265307345291283280277275276259263252295277252252261242272262272249274246205243256257235242250272224257242251273240241262231268239247236244251244247249255243254274266266275280231268279280268226304235248283241254264272249239275275254237266268239254259306275277269263278278271252255266260245248236230248261234248 079100200300400500600700800900>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.

4 205 921000000071 823 527000898 615 717000000000517 664 8090000588 491 61600001 277 501 00900002 588 027 13200016 896 825 84500510152025303540Phred quality score0G2G4G6G8G10G12G14G16G# 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).

99.7 %150 859 35299.7 %0.3 %

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.

99.5 %150 510 75099.5 %0.5 %

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%).

0.2 %348 6020.2 %99.8 %

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 %75 639 58850 %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.

98.1 %148 440 91098.1 %1.9 %

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.

10.3 %15 550 75810.3 %89.7 %

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.

5 684 810126 83776 030153 679110 719118 509135 048182 33881 086143 26960 36454 39678 88089 07244 538108 52068 03577 881108 973149 614152 701149 927179 095143 219232 981406 90922 752754 34733 42433 56572 89671 47732 68888 46834 81034 73059 13778 76120 037120 7091 948 31684 89476 538133 911112 397211 401189 646279 329517 93350 93672 09759 02682 02735 84764 04169 47348 494207 50250 163107 843136 948 180051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M120M130M# 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.

99.76%99.76%99.76%99.77%99.76%99.76%99.77%99.76%99.76%99.76%99.76%99.76%99.76%99.76%99.76%99.79%99.77%99.76%99.78%99.75%99.76%99.76%99.63%99.71%0.24%0.24%0.24%0.23%0.24%0.24%0.23%0.24%0.24%0.24%0.24%0.24%0.24%0.24%0.24%0.21%0.23%0.24%0.22%0.25%0.24%0.24%0.37%0.29%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped