European Genome-Phenome Archive

File Quality

File InformationEGAF00002308732

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.

67 665 748115 164 778164 552 616211 368 504249 595 822275 154 715284 646 158277 639 511256 304 257224 879 335188 122 364150 684 998115 947 73685 988 93861 764 79743 054 94729 253 93819 479 91612 774 8748 298 7965 371 7233 515 6192 350 3071 619 6371 165 092883 465696 921573 695491 595430 164382 353343 380309 240280 482252 498231 195211 481193 703178 226165 440156 063144 534135 255127 074120 762113 308106 782102 04796 96091 11186 83782 26078 71273 34170 02165 90863 86461 05857 77553 80250 95548 12946 56744 19043 40341 25539 65738 07936 14234 99733 74532 37431 03129 95028 34827 85926 88326 21225 67124 97324 45323 65722 81121 96721 08520 98920 20820 10319 92319 08118 36717 74617 32516 72716 10115 86915 50415 35915 19514 94114 44413 99613 74713 61513 38312 73012 59212 37712 07411 79511 29711 26111 22210 92910 58010 46710 3849 9069 8859 6499 5559 4719 3738 8939 0378 5968 6858 8268 6318 3508 2618 2858 0197 7707 6187 1887 1377 1647 0317 0636 7586 6936 7666 8256 7486 6556 6376 4436 2606 2916 2315 9475 8545 8995 6945 7725 6015 4925 4375 6435 4795 3645 4995 4395 3225 5435 3045 1535 1125 2235 1415 1794 9934 9734 7384 5494 4974 5404 4784 3694 2634 3914 2504 2234 2784 3464 2714 1264 0924 0404 0283 8133 8583 8083 9573 8263 7343 7543 8953 8053 7873 4633 4253 4743 4253 4883 5383 6883 4553 5323 3933 2613 2763 2963 4173 1443 2683 1893 1863 1802 9683 0173 1793 0322 9702 9212 9702 9422 8812 8832 7682 8072 8262 7652 8152 7832 6812 6972 7222 5292 6832 7172 5932 7352 7412 5252 5562 4892 5102 3852 4002 2972 3562 1902 2692 3052 2402 4452 2012 2042 2632 2352 2772 1422 2692 1402 2392 2402 1972 0912 2352 0592 1062 0911 9641 9612 0091 9371 9061 9361 9062 0231 9391 9031 8181 9101 8701 8161 8171 8211 8281 8501 6861 8281 8101 8671 8011 7881 7981 6981 6631 6231 6011 7001 6721 6541 7461 7251 6971 6611 6741 6901 6511 6971 6521 7051 6131 5451 5881 5771 5301 5171 5591 5311 5311 5681 5871 4701 4961 4381 4461 4201 4881 4331 3951 4701 3881 4451 4611 4201 4571 5421 3901 4901 3661 3991 3951 3581 3061 3941 3031 3151 2931 2881 3591 3631 3091 3171 3421 2991 2521 2281 2991 3401 3251 2771 2651 2701 3711 2411 2031 2041 2321 2291 2271 1371 1541 1821 2441 1541 1281 1601 2091 1841 1871 1301 1731 1511 1621 1541 1021 1061 1211 1031 1031 0571 1011 0571 1249921 0279841 0971 0771 0149829619459601 000939988951916991946924888942922887915903882909894910934915919901870848833817835816828856836762847770791802799795774879804749806779789862805744762789763778786795771784805759716709754735718738736709659752706690729741680633704701660723691657658679650719672697706656667665601674704676594611641647650606607630611654644643666650660642656650696634731653622643606648627611660625648593599550581618621597560584604543598623610563563559552628566573502559515559544517518567503526549543547533555585533564551539508530520506529589525480524542516536534545495502543509544496518504490524471497503485508491421492467512435450469466455465453450466479437446432447428482460473491461475450471469431481494486498493442431453409449403482450441484438420400407406371462429390403391429411411411413372370413381395399380394376393364393362393361410409379378416350408418377423430415410452378383390355397396400348386400368415410392398401426431362406379371433417403399377383409391349396409404397539449476432443410394381381393390421397395391407410376402391385368389406417397405394376409410431433439403408442429396433380397367386386383381385380411333405357363385381381359352376366373354375409379366375381352366325325355353341376318324343357369348364349359359351344359348367337376395388368381321364379360343367356374364386389359357355391354331378347348373358373386385385442387372360358333350308364381331327359350361335332373347367363389380365362345387329347357378319329337346355364377348329349390334334326347316376333385342374387417405377405369391338344375369383324322367345334367325310333378366326350338376349367341350338341329311329312336337307311309319307287295332300283288309300315310309311320264312271322273272290343308299310323320296311302303273263278335279317299291306275265252304273265273303295297281273288 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.

5 090 069000000039 515 329000882 837 805000000000504 077 6240000559 516 96800001 278 953 38000002 641 779 75400018 493 673 22900510152025303540Phred 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).

99.3 %160 453 18399.3 %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.

99 %160 027 87899 %1 %

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%).

0.3 %425 3050.3 %99.7 %

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 %80 812 72950 %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.

95.9 %154 997 95695.9 %4.1 %

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.

9.2 %14 900 2879.2 %90.8 %

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.

8 468 458146 49292 263176 940132 169142 975159 740217 650113 639168 64281 98269 05295 057103 34655 155123 13087 57497 799124 365170 432180 969179 471232 460172 063280 016456 15433 649781 36345 30441 84784 53787 64549 992106 41445 21345 17770 59691 92328 547140 3191 906 538102 41398 544168 562137 272253 058219 643327 985555 35267 16587 34175 24998 10947 580102 70084 71065 963216 58263 245128 065145 906 248051015202530354045505560Phred 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.

99.73%99.72%99.73%99.74%99.74%99.74%99.74%99.74%99.73%99.73%99.73%99.73%99.74%99.73%99.73%99.74%99.72%99.73%99.73%99.72%99.73%99.73%99.82%99.69%0.27%0.28%0.27%0.26%0.26%0.26%0.26%0.26%0.27%0.27%0.27%0.27%0.26%0.27%0.27%0.26%0.28%0.27%0.27%0.28%0.27%0.27%0.18%0.31%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped