European Genome-Phenome Archive

File Quality

File InformationEGAF00000644691

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.

207 197 47940 104 15710 447 9756 346 2044 583 3753 852 9843 365 2113 024 6142 755 3242 533 9912 365 2802 223 2112 098 8681 992 5121 903 6371 818 6861 742 4261 676 5151 614 2351 558 4881 503 2931 455 1811 405 7831 357 4171 316 6441 275 4831 241 1221 200 8491 162 8481 130 9521 096 3251 061 8611 031 760996 403965 678936 330909 767880 403850 478825 711800 655776 889751 414726 909706 327682 939661 362640 404622 468601 266580 232563 303548 342529 113511 916496 260479 615464 924451 781435 627422 200408 982395 961384 758373 399358 883347 606336 444323 592316 866303 834295 077285 769276 503268 075259 224252 047244 578237 686230 856223 262217 270210 267204 278198 129191 904186 042180 669175 858171 250165 740161 134156 934151 847147 556143 598139 217136 256132 090127 997124 809121 367117 284114 892111 333107 827104 402101 822100 09497 33894 61292 74189 82887 87685 08682 95480 79978 64976 61274 27772 64471 06169 21167 22265 55563 45662 23360 51758 84557 39955 91854 63053 39752 34750 55149 16447 91746 88445 80644 41143 51242 55140 79440 20638 88438 33037 34336 33235 89235 22733 95833 07632 11531 52030 89130 55229 49128 71828 19127 88627 16426 56126 02825 43124 72324 39823 67223 04622 74622 51422 10921 61721 01420 48320 25619 76719 14018 69918 61718 48017 98817 81817 53517 04116 66116 61416 26515 90015 39215 50214 91014 64414 47514 19013 84913 60313 29912 72212 82912 39412 26211 94411 84711 52911 45011 30410 97410 95110 77710 61910 33010 1819 9729 9329 8639 5349 5479 3079 2708 9718 8508 6728 6538 3628 2347 9147 9527 7407 5857 5117 2717 3947 1066 9736 8456 8266 6766 6056 5656 3206 2136 1326 0455 9485 9135 8055 5935 4655 4625 4195 3695 2115 1605 1105 1125 0434 8834 8664 8654 7654 6544 5154 5254 3374 4474 3004 3084 2404 1794 0803 9524 0633 9533 9723 9083 9623 8033 8993 6833 6073 4933 4643 4523 5233 2833 2843 2093 2563 1663 1283 0863 0772 9662 9832 8152 8322 7612 8032 8652 7152 6972 7902 7582 6132 5722 6082 5182 3812 4522 3542 3322 3462 2642 2232 1702 1662 1812 1712 1412 1082 0802 0362 0761 9981 8951 9041 8351 8331 8131 8631 7611 7561 6631 7771 7201 6271 7491 6641 6251 6691 5851 6031 5701 5501 5351 5211 5031 5091 5351 5381 5101 5141 4161 4781 5091 4281 4171 4051 3961 3731 4261 3121 3781 2751 3641 3511 2761 2671 2631 2911 1781 2041 1921 1771 1461 1841 1211 0991 0631 0981 0461 0471 0161 0751 0519901 0401 0061 0051 0801 0019721 0329919879909901 0101 006983946916921898896892917888875815829816814850802797810764742799778755771748707735745722732706724763709689694700691703608699674657663695661679662699674688675674699690711643681687656641628682598669623645631657587623587564630607556623526561526550526555571549550525494483554495558514539492535499470560512485514447455493508439445447448452456413503426449456477449415452432398465440469460414463439455414411412403470431439412411412425403405446450425431378398397399393375384361399384372359357352381353338350369360346325365342348317361341340326315333322314294324317332307328318316286327334336288313326318301275284288297302283302287276276310282260258322298273271273286242266234302282263244265280290257284285274260253253245264238255278272273250276262256267326271294274250272254236250242256255252270269246261237250219246234223211238223231216228228231229246228202225223226224216230216246241237224244217205199193214206210192207217213218201188193206181161173179179162172169178169164136154166197157139151139125167150145144125142136130152151158143142134121130126125136129133144133123131114143146129137134151117126119126127133142113117123130115117121114999310695919783928810492107891038484998810081928784828378728681546880747278727393646564747872727762828167797159737774657864727677777467726475637579677369557563645958486157606767626064666760524865576555545551595158655542524772514751454552455259654363486956464944415442495254413139485138475338424447353841424431353238463440373441312945313333262432312230203126302828302230313027283428253225292629323041292821262131292925283429292326302627433430272136253911 180100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# 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.

186 19991 791190 832177 340583 841391 6201 424 9913 835 0141 457 9353 868 43310 216 1743 391 4862 757 7495 095 9822 259 6392 390 0672 144 6721 428 42112 389 57618 957 37914 004 79410 884 9224 245 0947 274 65010 621 88424 430 71930 290 57926 540 75031 433 30832 609 32243 396 18262 582 41890 726 161109 155 345179 876 459242 770 180331 123 531497 434 170829 594 909796 629 545206 298 54334 160 14616 374 2872 144 08901 001 2720051015202530354045Phred quality score0M100M200M300M400M500M600M700M800M# 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).

99.4 %49 145 08899.4 %0.6 %

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.

99.1 %49 018 28699.1 %0.9 %

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%).

0.3 %126 8020.3 %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 %24 725 61650 %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.

98.9 %48 924 50498.9 %1.1 %

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.

4.8 %2 365 4154.8 %95.2 %

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.

5 299 7443 7933 8127 5123 89811 1259 99521 47918 10547 63042 45412 28492 89617 03615 978206 20835 68680 90776 25330 303155 0021 42799 835341 8351 15114 0252 0591 9391 9331 103 1055 1284 2965 2206 6387 06010 374210 390637 55418 5484 65434 85217 9841 96647 8142 0023 306141 2183 2985 9465 67613 9947 64025 45825 01241 35676 592184 63440 143 213051015202530354045505560Phred quality score5M10M15M20M25M30M35M40M# 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.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped