European Genome-Phenome Archive

File Quality

File InformationEGAF00004837052

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.

234 151 779397 159 067495 061 193495 693 207418 467 513307 889 496202 137 256120 699 66666 686 05634 700 71717 311 2308 543 4244 294 1532 324 3161 401 427948 775716 143576 752479 814405 004356 748312 235276 999245 379218 062192 053172 464153 535135 247122 083109 99199 43888 50080 25072 79666 82361 65057 09451 52349 82847 90545 19242 74539 48337 07335 37433 71431 75630 91029 45427 98227 13025 59225 11723 78322 84822 69621 55721 48720 75119 58119 28018 67818 36717 16216 82016 63616 59216 16015 27814 84615 03913 85613 95013 60213 10613 04712 70412 53012 08012 12811 70511 55111 44410 89410 64710 61710 0709 9639 8519 5229 0058 8038 7618 6238 7288 3207 9347 9138 2077 7067 6497 3967 2507 2767 3307 0097 0147 0136 7946 6766 8106 4986 4516 1746 2356 1946 1756 0356 1195 7365 5885 5195 3865 1495 1285 0955 1254 8984 9235 0064 8684 9604 7524 7514 8224 4824 5044 5944 3194 0234 0784 1143 9893 9433 9963 7143 7603 8193 6203 7773 5383 5643 4713 5243 5193 2553 2683 1513 1673 2353 2673 1163 2013 1673 1592 9202 9862 9932 9562 8642 9712 9883 0793 0763 0152 8763 0432 9372 8312 8452 7432 6872 8382 7732 7232 6462 6572 5812 4552 4942 5322 3872 3462 4332 2872 3292 3022 1892 2222 2552 3352 3652 2382 3342 3122 1662 2412 1382 1782 1192 1202 0972 0622 0772 0752 0321 9781 9551 9741 8281 8691 9001 8561 7901 7851 7491 7301 7061 7291 7441 7191 7961 6851 7281 7171 6771 7271 6981 6581 6721 5751 6671 6441 5371 6181 6611 6831 6631 7531 5901 5661 5681 5721 4631 5651 4681 4791 4701 4221 5191 4741 5501 5511 4931 4861 4351 4581 4921 4661 4081 3671 3901 3361 3941 3341 4341 3711 4251 3421 2721 3311 3571 3201 4101 3891 3331 4241 3211 2531 3311 2801 2471 2821 2431 2581 2441 3721 2671 2461 2881 2191 1761 3271 2271 0981 1351 1101 0741 1261 0871 0921 1041 1101 1831 1321 1341 1211 0701 0281 1911 1861 1091 0791 1121 1081 1381 1391 1511 0561 1731 1781 1031 0721 0711 0731 0271 0339429961 0179439921 0209741 0111 0271 0321 0131 0581 0631 0331 0531 0151 0841 0969971 0851 0611 0971 0101 0831 0291 1451 1481 1151 1081 1031 1711 1161 0601 0521 0951 0881 1301 0391 0709971 0809411 0059681 0261 0191 0431 0791 0501 0651 1101 0561 0291 0511 0631 0481 0861 0761 0341 0731 0281 0749809629871 0431 0511 0101 0039829581 0069241 036971975941931979956980898936950932948962983909906916911847855886832826773776809867853921785803816833700737819764770772799800725756732714737772647707743692718702706703704736789677710676672681691662674697661716657647664644646680628629618634619630619613656597638659586599572606602552545522571583559545549524468524558530563547535536532549566519527515487483478516521477480498470466486458463475464447438461414447412430411449420425379386374398389364400415407422415382431389410384415395418412414380405382368378390436412428430411390357406428394413429391421412373434386420369384416399404414414390375345412345337342364352362392378364382366339396329350374372373366342334395356321335313363333316357333343329332301333330319292306302300324315326347316320339292317324331316339325318318286363337328362299311298312296317283316314335292281322279306287273316335311318354355320323318294327316305284290295308265296273295283294261282257269275277270265254270259236258247253256263247256272257252250259291244285253255258284231249245265246277253250231256249223253260238251232228255247258202243269236216265248224240249292224232233277257271242244253242256219263227231256234232249251248260265233226252238230237235250226237242251234240254232282254274235234262234263263255266253249259251260243252274263274282263272269243237261252232241237249248290240260266265265276259263249252270261275270272304258260244276239230267247232252232270265209280225249273271279263276283243251256244257256271260237224237235217253227208241225258238245261271233256247227253261251234257260255241236253263232279244256248239231239251250232313278237273284223248226239270242251247214243250238251213253255215226229213223198244255226216230230246213239240236225195219243223242204188230231229214232232238208256257246196216247233218157 812100200300400500600700800900>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.

590 0600000000142 710 1860001 228 824 487000000000664 036 4190000666 506 62600001 137 590 99400002 037 620 2850007 160 860 71300510152025303540Phred quality score0G1G2G3G4G5G6G7G# 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).

99.3 %85 780 21699.3 %0.7 %

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.

98.7 %85 258 33298.7 %1.3 %

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%).

0.6 %521 8840.6 %99.4 %

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 %43 174 63550 %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.

97.5 %84 157 75697.5 %2.5 %

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.

5.5 %4 774 7845.5 %94.5 %

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.

4 169 63187 54956 458102 50678 95879 59099 416118 12452 63993 05545 71539 35162 00460 75532 08170 00252 20159 68481 57098 68199 65296 255115 87590 519149 429233 45315 618424 83223 72222 54049 85545 41220 49356 08923 64823 27544 78451 18514 28475 7001 270 67457 13463 18186 88679 612137 177117 768185 905233 99443 08246 89847 02752 35036 23460 52558 46349 034127 01855 68487 01776 513 636051015202530354045505560Phred 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.

99.37%99.37%99.37%99.38%99.37%99.37%99.38%99.37%99.36%99.35%99.36%99.37%99.38%99.37%99.36%99.41%99.38%99.36%99.42%99.34%99.38%99.38%99.6%99.16%0.63%0.63%0.63%0.62%0.63%0.63%0.62%0.63%0.64%0.65%0.64%0.63%0.62%0.63%0.64%0.59%0.62%0.64%0.58%0.66%0.62%0.62%0.4%0.84%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped