European Genome-Phenome Archive

File Quality

File InformationEGAF00001703907

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.

107 550 90232 994 56913 745 0017 948 8765 331 5223 947 5573 122 8732 578 9172 222 0051 940 9531 724 3471 561 2211 428 3431 309 1851 215 0961 133 9361 057 437996 517941 820889 786844 594802 385763 567726 597692 527659 618633 282607 229585 181562 458540 322521 051500 624483 784467 499450 237437 106420 939407 220393 834381 012368 470357 168349 161336 884327 458315 856308 261300 601292 334283 792274 125267 142258 886253 367246 629240 299233 987227 800222 168216 345209 970206 413201 587195 829190 236186 214182 093179 097173 917169 623166 041162 378158 728155 994152 610150 300145 571143 016139 957138 585135 898131 686129 405126 607123 125121 366119 158116 369114 551113 035110 462108 847105 329103 320102 04099 88296 75396 02193 35292 07289 81389 21987 24385 23683 93982 20980 45180 08878 36177 14075 82274 22473 75672 18970 29469 26668 86066 80665 34364 54063 11662 22961 52160 38459 53158 39157 65056 32655 03254 69753 98853 28752 06951 56751 07350 39648 89349 00148 50347 80646 52845 94046 05344 33543 43243 05243 18341 77140 63040 25240 28839 20639 21637 92537 82037 56736 82036 69736 10335 60435 34035 19034 15634 03233 47433 14132 48132 12031 08630 98330 29430 34830 23529 54329 47528 73428 36527 89927 66727 74327 44126 94226 48526 25325 70824 93825 10724 98225 11224 25223 85623 89123 33322 96022 91522 50622 12822 39021 97221 37521 48021 15020 89320 85520 60120 06820 29120 31719 92319 49819 57118 75519 24118 57117 84317 71917 94917 52717 27117 25917 59316 92816 96316 51716 51416 38316 13816 03115 80015 91815 71315 66115 19015 37315 30614 80814 69714 61514 44914 38714 08813 92514 01313 87413 81113 73913 51013 63213 70213 55913 26412 87513 09812 73012 44912 55512 37312 68312 14012 25011 81311 93412 09711 70511 33911 55811 49611 49311 57211 14211 02111 03110 94010 68411 11810 51310 62410 49310 49610 57910 04810 1209 8329 86210 0149 6039 5309 4209 2579 3539 2419 2089 0808 8509 0209 1568 7678 6218 7168 6588 5778 5328 4048 5298 4468 5638 2988 2368 0378 1858 2167 9317 9337 8047 8257 8417 6337 5027 4807 3437 4257 5517 4077 5777 3647 2847 2977 1807 2327 0776 9107 0646 9486 8606 9166 7656 7926 7166 6786 6906 4956 4886 5336 5386 3426 4036 2556 0276 0446 1306 1155 8766 2485 9646 0326 0465 6675 8276 0405 6335 8715 6075 8485 5385 7015 5605 5385 4655 3305 2715 3375 2115 2535 1185 1945 0495 0975 0545 1015 1075 1955 0285 0354 8954 8405 0864 8564 6704 6424 6924 6424 7254 8124 5584 6734 5244 5384 7904 6914 7704 4684 3864 7254 3874 3074 3244 5834 1994 3914 2184 3714 3124 3924 4264 3334 1044 2844 3594 3564 3644 2724 1424 1424 1113 9804 1124 1133 9763 9914 0214 0893 9074 1713 8813 8753 8023 8923 9093 7503 7983 6423 7083 7813 5933 6103 6933 6203 5233 5133 5733 6543 5013 5083 5043 5253 4053 5613 5633 5683 6053 4883 3583 3623 5533 5033 3383 1813 1873 2153 4803 2293 2553 3183 2343 2913 1433 1653 1023 0453 0883 2383 1903 1143 0283 1043 1643 0033 1723 1882 9003 0243 0422 9652 9162 9292 9752 9852 8972 8042 9492 8642 8212 6202 8242 8632 7942 8642 6792 8222 6972 7512 6912 6202 7392 7042 6662 5302 6082 6072 5642 5512 5762 5672 5472 5572 5412 4222 5542 6082 6102 3432 4822 5072 4402 5402 4482 4232 4582 3622 4152 5042 3692 3962 4282 4452 2992 3102 4952 2952 2652 2862 2922 1882 2502 2842 2302 1902 1852 1242 2622 1532 1962 1172 1672 0902 0131 9682 0322 0342 0522 0001 9782 0712 0602 1632 0242 1011 9891 9122 0401 9102 0111 9201 9622 0711 9801 9251 9251 9591 8961 8201 9101 8201 8891 8741 8091 7401 8721 7891 8491 7821 8791 7381 9391 7861 6841 7901 7181 7701 7331 7401 7901 6561 7251 6961 7471 7651 7641 7021 7081 7121 6951 6731 6251 6491 7411 5761 5361 5671 5781 5801 6771 5251 5241 5131 5751 5641 5151 4281 5421 5201 5461 4781 5791 5371 3661 5121 4671 4191 3931 4851 4201 4681 5131 4091 3771 4711 4731 5051 4451 4341 3491 4321 4021 4581 4281 3041 4061 3491 4331 3331 2761 3811 3461 3301 3821 4491 3841 2731 2781 2811 2821 3141 2881 3421 3091 3171 2091 2021 2211 2791 2061 2451 3001 2761 2601 2581 2181 2101 2221 0961 1201 1491 1421 2301 1281 1601 1231 1281 1061 0701 1401 1001 0521 1321 0861 1231 1251 1311 1421 0791 1091 1671 1071 1261 0591 0581 1391 0641 1171 1421 0401 1199581 0971 0191 0521 1171 0151 0281 0831 0011 0191 1071 0201 0991 0221 1321 0841 0909899521 0751 1151 0829941 0131 0171 0789819951 0081 0099909959671 0099899539839559529819389211 0091 006926980956947874907893984857952891891886931870891828852952934883861796883872853858843871932846777857836853825835954804805830831831765824769799730807793808796825718780807746740786802823726704734730787772811770705773734751747752750793777796816683745757658692708727744819754732733769701750726646709683658650675680702700702682649646728642689652609628636708645629669632621647626564664607618597651633639585610598605661673628650635630587584604632567602634670596598598578602661626635569579599537599600565603582633597592587571590553580607582569527540535572558512550573546539601535556548523503479506547510496515570523507514491492490510479545496536504489487511563504492531502551352 152100200300400500600700800900>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.

4 294 4010000000000000128 649 93800000008 395 5650000112 864 18400000433 941 8230003 525 260 78900000510152025303540Phred quality score0G0.5G1G1.5G2G2.5G3G3.5G# 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).

99.7 %56 030 97599.7 %0.3 %

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.

99.5 %55 891 48699.5 %0.5 %

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 %139 4890.2 %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 %28 089 37850 %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.

82.6 %46 404 27282.6 %17.4 %

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.

12.8 %7 175 54112.8 %87.2 %

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.

3 691 232124 47528 092215 507247 33980 102117 606153 91130 916264 33376 28164 837184 460111 80937 889197 50538 07152 66669 965104 42528 786193 09982 898115 679200 152354 16042 376772 35345 616157 63662 12585 63935 021210 69125 97252 44666 349121 10836 645284 2971 881 36178 85570 620110 053181 844140 03387 482103 972114 001208 663196 113221 779913 69550 087177 676111 15845 390271 330141 92629 48744 598 071051015202530354045505560Phred 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.

99.77%99.77%99.77%99.76%99.74%99.73%99.76%99.77%99.78%99.78%99.76%99.76%99.76%99.76%99.76%99.76%99.75%99.78%99.75%99.77%99.75%99.77%99.75%99.83%0.23%0.23%0.23%0.24%0.26%0.27%0.24%0.23%0.22%0.22%0.24%0.24%0.24%0.24%0.24%0.24%0.25%0.22%0.25%0.23%0.25%0.23%0.25%0.17%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped