European Genome-Phenome Archive

File Quality

File InformationEGAF00004837423

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.

310 718 354405 370 181420 214 246391 483 596328 105 270258 380 187191 199 340136 002 56493 095 92362 019 02740 231 60525 676 90616 163 88610 129 8386 335 1263 971 7932 513 9181 632 2821 090 186759 398552 460424 360333 791273 180229 399195 647169 723152 416132 915121 464108 91598 99990 30182 32675 30469 49664 60559 72755 49252 42247 51344 52442 35439 82837 56036 32234 26731 87130 91729 56727 38926 59325 24424 10522 73921 86321 60920 26920 03519 40818 19817 16016 77015 92515 42215 27414 41313 76113 87113 55213 26212 80812 12511 74811 62511 39611 04110 64610 39410 04010 35110 0059 9609 6529 4388 9569 0058 9678 7958 4598 0728 0027 9197 6837 4327 4477 4706 9486 9206 8306 6586 6286 7906 5976 4176 2555 9415 6025 6275 9925 9795 4905 4865 3645 2425 0685 1255 1394 9144 5904 6414 6574 4994 4454 4164 3724 2034 2164 1014 0664 1423 9263 8363 6613 8213 8323 8223 5573 5663 7193 5703 4923 4443 4333 4203 4323 3533 2013 0863 1783 1463 1243 0953 1803 1102 9042 8862 9422 8272 7572 6692 6952 7002 5602 6512 5892 5852 5102 4952 4352 3242 4342 5012 5082 2982 2082 3072 1242 2652 1802 1002 0862 0382 0332 0152 0651 9441 9211 9431 8961 9021 8911 7721 7841 8621 8941 8241 8121 7361 8511 6611 6221 6361 7051 6421 6071 5991 5541 6431 6201 5681 5741 5281 5251 5691 4931 5701 4891 4221 4561 4301 4191 4691 4701 4151 4061 4101 3971 3481 3271 3611 3711 3331 1931 2571 2501 2521 2491 2471 2801 4041 2731 2891 2511 2611 2821 3161 2311 1931 2171 1311 2141 1771 2011 1691 2261 1701 1731 1691 1921 1091 1601 1001 1511 1251 1391 1081 1431 1091 0451 1331 1131 1321 1091 0831 1841 1301 1471 1691 2071 0261 1721 1891 0901 1501 0909831 0159811 0091 0589559399959829949779599829989661 0281 0239969851 0519878909698959659489429909019439189729439509709729179421 0089149058839619541 1121 1011 0069929399389598539099811 0059561 0219609619509519049509819079369339059239899521 0079379989801 0451 0081 0349831 013992937978886958895897901878905907923926793803798803837785789817773774786739757819813784842772772796829767789768820797765770788815845856803754640759692701728703714701680707713774705755764705745738685655717670683683670685644659643640652689578586595554573551571533573516542515564576512505542512511478488486473529565525489510486473436471444497477470448430425438423416427449453420469429432396425374424373402377413410407367379386381412383372357394360325380369356381320329362337386344337494335342355308330340353309316337328323337337333347346329331335327356378326352338287271307280274303295281280317300305287306299302309310289317308278297268295308271338298301298295261249245264266268259257258311268254272274278290291239304275288279260286273275298266288305315296331314329313309460303319269281274300334305252257301239249272237229245238242266272228296282253286310253280299256289291267262286285244259261259319295273266238282267312311278298307314294283293282290280261268253236279233263260233269265260281274277275241271270284278240271286268229289265275276286241257249227259261244252233256244256214233222232232247208202209233234222226247244221247222225248230238225196231248245249204228228240293245276262237202240250238241241238249255257240275266263251256234223263252250264236253252246238261268248256267259236264267270271244250246228264258241276286231252224271238255269226249224261249260239242269251273258255281289255272252262248237262272284231253230227231212238226222209229253221217240228232239255233225206196230236223228237217224229225241241247247244258261223241226234261237245258232244216223259222190212229227251265227191186224216182215190198209205182203194190221186202189208187216226199213209204213221216210185215173192186188201206184201177165205174189185209184187173164185198193159194194173184164194194138181161176159151168171170191141163177164158163149158144182151175170164173128156141146142147169153157139172174123 996100200300400500600700800900>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.

1 635 916000000079 147 173000730 752 600000000000403 713 6860000447 428 2200000874 792 72500001 730 971 4020008 752 813 45600510152025303540Phred quality score0G1G2G3G4G5G6G7G8G# 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).

97.3 %83 946 04497.3 %2.7 %

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.

97.1 %83 741 28097.1 %2.9 %

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 %204 7640.2 %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 %43 116 73950 %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.

93.7 %80 828 91293.7 %6.3 %

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.

16.5 %14 250 92016.5 %83.5 %

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.

6 096 82167 62946 29782 69263 10267 37476 44998 97659 33881 69541 16134 71343 02946 44625 56255 77338 69441 50552 50070 76770 62480 717102 60879 717126 350200 81019 214361 76623 93721 41538 55440 81725 66151 86122 49323 41132 98241 86015 74366 642789 31650 00051 07882 28068 847124 953112 204169 128274 81438 46145 51740 70350 95128 05161 19845 58937 137107 21338 82670 66177 728 502051015202530354045505560Phred quality score10M20M30M40M50M60M70M# 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%99.7%99.76%99.76%99.76%99.76%99.77%99.77%99.76%99.77%99.76%99.76%99.76%99.76%99.77%99.78%99.78%99.76%99.78%99.77%99.76%99.74%99.8%99.69%0.23%0.3%0.24%0.24%0.24%0.24%0.23%0.23%0.24%0.23%0.24%0.24%0.24%0.24%0.23%0.22%0.22%0.24%0.22%0.23%0.24%0.26%0.2%0.31%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped