European Genome-Phenome Archive

File Quality

File InformationEGAF00004841314

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.

32 049 32270 074 611125 374 628192 653 339260 433 106313 757 170340 411 283336 086 231305 178 340256 759 706202 032 876149 519 531104 882 88470 157 20244 996 35227 977 98416 929 18610 106 5026 057 9173 712 2472 375 2711 608 2131 165 304907 044738 955629 949546 736480 934424 654375 981336 040303 136275 634251 004229 168210 722197 271185 378174 151162 974152 653142 253134 957126 759119 691114 580106 663100 12295 37890 18085 93779 61573 76469 85866 44462 13058 06355 15652 63949 53047 95344 46442 63240 83138 82337 52435 74234 01333 49332 59831 27430 21229 11728 29827 59426 67026 47125 16624 20424 11623 77422 54021 86421 23820 82820 11819 91519 61018 63918 49118 04317 86917 37617 17016 66916 38516 26515 77415 14914 41214 07114 24413 86813 52913 21113 05613 05612 98812 80812 59412 02611 87411 77611 61611 19211 02210 99710 97610 65710 27710 32110 1449 6919 6049 5579 2789 1089 0969 0309 1909 0389 0478 7268 6008 4908 3088 2148 2747 9748 0627 6787 6127 6167 3327 2147 3007 3097 1157 1067 0477 0176 9396 8656 7826 6236 7176 3166 5336 4806 5946 4016 2676 1125 9246 0115 7145 7505 7915 5955 6225 5855 6285 4895 4375 2495 3515 0975 0955 1164 9794 8825 0414 7304 8454 8034 7554 6904 8194 8084 7494 7524 4914 5004 5434 2854 3374 3434 3794 2394 1574 1354 3034 1914 0384 2444 3094 1324 1353 9233 8723 9123 9603 7993 8923 9003 6623 9593 8763 7473 6183 6463 4083 5543 5743 5673 4983 4683 3983 3573 2453 2823 4653 2783 2563 1553 1333 2833 1383 0493 0102 9932 8942 8842 8682 6822 7362 7522 7652 6422 6082 7572 6942 6712 6242 7192 7932 7292 6982 6212 5302 6182 6302 5842 5312 4162 3672 4592 4252 4212 3532 5852 4962 3142 3802 2362 2582 2612 3032 2772 2182 1792 1011 9962 0322 0842 0632 0792 1092 0802 0882 1012 1061 9942 0562 0811 9391 9971 9541 9782 0251 9011 9411 9891 9512 0161 9311 9011 8641 7571 7761 8331 7081 6791 6231 7871 7611 7111 6741 6671 5641 6551 6651 5511 6461 6941 6971 6741 6701 5751 6031 6041 6401 6171 5771 6351 5691 6271 5511 5331 5511 5101 5251 5221 5391 4081 5041 4621 4451 4331 4571 4731 5301 5821 5211 4011 5211 3551 4831 4611 4821 4481 5091 4381 4101 4411 4011 4061 2921 3681 3361 3051 3001 3941 3011 2931 3961 3451 2541 3391 2491 2731 2601 2481 3071 2631 2501 2861 1671 2801 2561 2701 3141 2861 1931 2491 2761 2321 2181 2121 1821 1961 1391 1831 2441 1551 1261 1261 1821 0881 1161 1141 0611 1071 1081 1271 1331 0991 1551 1281 0731 0671 0961 0611 0991 0941 0761 0521 1381 0211 0181 0481 0681 0161 0191 0711 0421 0721 0981 0891 0881 1061 0641 1141 1051 0399879841 0331 0359641 010983993924962967983939905998986925910892910901875915903883903844876899832836869840811795795827801804847904868840917859817803790826816790770767813774688710776731746735746726793715697764782869849705718733699690717735692708667691679696653681707664672659648616609655643621610625623600666605615649656609649654579646633661637623578623683650661646632637619592599593575641610588623596565560585571536584554603570554567615571589544543562517553554646541545529548547545514539536530510491516502542508512527547493527509528511532542492502486513511486487488479495457491488437462431462448438430447432451469463413460442404448464471459446432459477498467450491491442412442424464432405450425467435384428402422487432400417424417430442448402412411442383469438388354390417416403370379406422394449425434439441402409416389378407413405381399404418422410406395364343372434421402393442432443430416463432434411380355416388400379426383430420404402368372391332338323377323350358369372351361327349376337368357354322357349320358354345338330330331399317368355323338338305310336327338337338320329326302336308318335319344343323344345350313314312310294302303298290298321312276287305314293274307273282312315274323310322310309269290274277271247294291283272288284285290281295308279300245258265267300273256239268268273253239248278269251275252256247242270252254275236309308282280253247250266250247217250236260247228264302276271255236244275248238256243247252228242262225228232242262261219259251260254246221264253243232226215245238224228240233220228210225205201208236208227244248212222210236216233227213227252231211216211208232212225190205219207219202217223208237237195209221186215224224222219329 760100200300400500600700800900>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.

2 876 762000000035 724 410000986 409 727000000000583 005 0710000652 429 61500001 461 485 83800003 080 921 44200018 131 631 87300510152025303540Phred quality score0G2G4G6G8G10G12G14G16G18G# 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).

99.6 %164 525 80599.6 %0.4 %

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.

99.4 %164 150 09099.4 %0.6 %

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 %375 7150.2 %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 %82 564 51950 %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.

97.4 %160 906 69897.4 %2.6 %

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.

7.4 %12 227 4617.4 %92.6 %

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.

7 906 593167 979106 440198 426153 773159 746177 058241 233110 599178 43486 11174 199103 144117 34162 486134 92699 006110 572146 466204 955228 453202 610257 594183 981285 865480 36631 434827 77146 01343 60689 95190 17243 298108 67546 86545 10874 91798 92827 038145 0852 158 72997 83191 500156 369130 888244 101210 872324 800534 94961 53681 32071 49592 71243 44178 85281 62258 024223 73760 718124 075147 274 034051015202530354045505560Phred quality score20M40M60M80M100M120M140M# 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.

99.77%99.75%99.76%99.76%99.76%99.76%99.77%99.77%99.76%99.76%99.76%99.76%99.75%99.77%99.77%99.79%99.79%99.75%99.81%99.76%99.76%99.76%99.83%99.69%0.23%0.25%0.24%0.24%0.24%0.24%0.23%0.23%0.24%0.24%0.24%0.24%0.25%0.23%0.23%0.21%0.21%0.25%0.19%0.24%0.24%0.24%0.17%0.31%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped