European Genome-Phenome Archive

File Quality

File InformationEGAF00006164709

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.

2 865 4943 635 5305 289 4318 158 60612 248 07017 501 70724 127 33532 243 42342 049 04753 926 37868 002 78184 250 670101 901 268120 186 872137 923 911153 649 120166 371 996174 919 632178 918 516178 249 828172 975 528163 919 881151 767 089137 406 757121 866 071105 966 04790 498 17075 939 72062 712 79650 991 94040 923 93332 372 70425 345 40519 596 56315 040 13511 440 4968 659 3986 540 9774 950 4153 751 3142 866 7122 211 1981 735 7951 382 1541 130 580941 663799 832692 459611 925547 846502 741461 625428 406400 806375 711355 891335 735317 115300 567285 928272 304260 207246 175233 858222 695212 587202 965193 158184 676175 897168 537160 489154 164147 778140 159134 119128 211123 327118 826113 891111 004106 935102 95399 15496 38493 44290 60086 71184 98383 28380 98379 36578 13076 75775 77473 80172 73071 76569 18067 37366 01664 04863 73062 00560 93359 43157 79456 23055 10754 61353 43151 42150 82549 02048 59147 05346 13244 96543 81442 54241 62440 26139 26237 65836 84436 13234 65434 16333 01131 94731 11630 26529 25428 55428 03126 99726 04225 25225 20624 64224 36223 55922 75122 48022 20021 20220 83520 55820 21319 70419 48919 03118 46318 28517 96117 18916 78516 52016 37915 79415 58815 64414 99414 50014 63414 37614 39013 91313 78913 86113 47413 27613 56513 00613 01412 61012 43812 22311 95112 04511 48611 77611 29811 33711 42111 05810 71410 49710 41510 17610 0429 5989 8349 8079 8029 2839 3819 3929 1789 1268 9498 7948 8438 5368 4448 1938 2448 1817 9357 9958 0707 8557 9387 9637 7747 4267 3437 4337 3437 2937 2617 0147 1687 0546 8876 7426 8346 7246 8076 8016 6216 6296 6426 7656 4896 3146 3716 2376 1866 1296 2076 0275 9835 6875 7285 6555 7295 6885 6875 3885 3435 5145 3725 2825 2745 2865 4235 2815 2965 0905 1785 2245 1185 1285 0125 0085 0034 9874 8264 7664 8274 7614 6964 6094 7054 6944 4604 4684 4714 2944 3884 2954 3494 2184 1754 2224 0064 0184 1443 9383 8323 9143 9843 9813 8413 8314 0284 0363 8643 8883 9243 8473 9343 7883 8683 8463 7763 6823 6453 6293 6573 4763 5983 6633 6333 6563 6233 5453 5333 4623 3253 4363 4983 3523 3293 2753 2943 3293 2383 3263 1963 1613 2213 1343 1913 0893 0503 0813 0633 0043 0643 0522 9553 0913 1132 9232 9402 9922 9062 9802 8613 0812 9972 9152 9222 9142 7852 7612 9612 7282 8432 7602 6892 6462 7262 8962 8702 7252 7712 6642 5982 7822 7482 7422 6272 6872 7132 7062 7302 6382 5142 6032 5262 4002 4422 4082 4712 5032 5112 2552 4152 2862 3402 3102 3242 3512 1572 3242 2052 2672 2602 3172 2092 2202 2442 1842 1912 1812 1132 0472 1832 1372 1512 1062 0952 1552 1042 0942 0872 1252 1392 1312 1982 0472 0002 0721 9732 1002 1572 0061 9282 0872 0562 0181 9882 0132 0252 0112 0171 9761 9472 0391 9572 0822 0531 9291 9051 8871 9171 9541 9031 8791 8941 8791 8431 8011 8411 8311 9001 9911 8131 9071 9341 8061 8991 8211 8051 8151 7161 7521 6921 6491 6771 6871 6621 6511 7371 7511 8051 7091 7001 7221 7341 6591 5811 8011 5701 6401 7671 7301 6871 6421 5771 7341 7271 6091 6871 5281 6021 5781 4621 4941 4531 5551 5411 6571 6351 5691 5771 5631 5431 4901 5501 5701 5641 4921 5811 4741 5931 4771 4801 4091 4471 4351 4851 5361 4311 4631 4371 4751 4071 4851 4511 4261 4351 3931 4231 4191 4931 4431 4121 4331 4661 3871 4481 3261 3881 4571 3881 4561 3931 3461 3651 3761 4151 3231 3101 2741 2281 3391 3091 3041 2891 2971 3621 3001 2761 2851 3411 3211 2791 2741 3201 2451 2071 3261 2881 2931 2401 2991 2311 2521 1921 1501 2361 1911 1881 1571 1611 1611 1661 1801 1361 1321 0761 0761 1371 0671 1391 1321 0891 0381 0991 1521 0281 0821 0511 0981 1081 1371 0621 0871 1141 0661 0571 0521 1051 0921 1291 0861 1151 1051 0881 0671 1751 1221 0841 1111 1441 1051 0791 1001 1231 0601 0659939929999869769631 0399711 0351 0201 0541 0191 0469701 0621 0421 0529289709619091 012940984920942940977903959974910918877954964904914971944969932891853926859903828843845856837881791826842810816810799824842839871824822818736741813846782803753786769772827853756794840763716712832752766731741744812737709690759737811775795791764780785745722798809805780749765802716707820749726723770781767816744805792746784752742728725769736699718737696653687683710675685724671718683665713687694698694662654685660677712643627668637711718731632673623653617654638628639599685659594647580602624583561562557571534571560616600615628583595532559606543604531543549582577544606541573613601600578561604609536580581601550583574577523504547602548513565556512500466518505538559517540532526608575530509525529522519532539488525530536529524580586580552540504543537527495523499490512518501481486498526504466510464473514523494515469483467494458445494455507485457488505503446460445472475469502467478473447467471454494447480456444465438434447493456440511497458505482470440472419444442419441442454482421450428431443437446436490443441436419439423428408391579 699100200300400500600700800900>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.

1 542 86100000000001 745 710 99300000000000002 867 687 3190000000000057 065 874 65100000510152025303540Phred quality score0G5G10G15G20G25G30G35G40G45G50G55G# 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).

99.9 %407 961 56699.9 %0.1 %

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.

99.8 %407 666 11299.8 %0.2 %

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%).

0.1 %295 4540.1 %99.9 %

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 %204 241 11250 %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.

97.2 %397 113 92497.2 %2.8 %

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.

21.4 %87 610 58621.4 %78.6 %

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.

17 300 502435 585279 366881 439401 522389 254754 613640 504275 209433 553192 525174 070220 079266 429295 506314 815221 961283 397308 311441 551478 674456 313589 190442 296688 2811 101 30171 9271 980 680104 38999 386193 806211 179105 137250 495107 693104 379160 252224 20060 730337 0084 842 367224 505196 683370 329299 606570 904510 609725 1391 394 024127 133187 679152 157213 68884 098168 759170 802111 619524 713111 206264 826368 508 311051015202530354045505560Phred quality score50M100M150M200M250M300M350M# 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.

99.93%99.91%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.93%99.92%99.92%99.93%99.92%99.94%99.83%0.07%0.09%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.07%0.08%0.08%0.07%0.08%0.06%0.17%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped